Compositions and Methods for Producing Tobacco Plants and Products Having Reduced or Eliminated Suckers

ABSTRACT

The present disclosure provides the identification of genes involved in sucker growth in tobacco. Also provided are promoters that are preferentially active in tobacco axillary buds. Also provided are modified tobacco plants comprising reduced or no sucker growth. Also provided are methods and compositions for producing modified tobacco plants comprising reduced or no sucker growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/307,035, filed Mar. 11, 2016, and U.S. Provisional PatentApplication No. 62/399,181, filed Sep. 23, 2016. All the foregoingapplications are incorporated by reference in their entirety herein.

FIELD

The present disclosure identifies axillary bud-specific promoters andgenes involved in sucker growth. Also provided are methods andcompositions related to reducing or eliminating suckers in tobaccoplants, their development via breeding or transgenic approaches, andproduction of tobacco products from those tobacco plants.

INCORPORATION OF SEQUENCE LISTING

A sequence listing contained in the file named “P34370US02_SL.TXT” whichis 729,504 bytes (measured in MS-Windows®) and created on Feb. 24, 2017,is filed electronically herewith and incorporated by reference in itsentirety.

BACKGROUND

Tobacco is a plant species that exhibits exceptionally strong apicaldominance. Molecular signals from the shoot apical meristem (SAM)mediate a hormonal signal that effectively inhibits axillary bud growth.Upon removal of the SAM (also known as “topping”), physiological andmolecular changes occur, enabling the growth of new shoots (or“suckers”) from axillary meristems (buds). Sucker growth results in lossof yield and leaf quality. Suckers have been controlled by manualremoval and through the application of chemicals. Maleic hydrazide andflumetralin are routinely used on topped plants to inhibit axillary budgrowth (“suckering”). However, labor and chemical agents to controlsuckers are very expensive. Control of suckering in tobacco throughconventional breeding, mutation breeding, and transgenic approaches havebeen a major objective for several decades but, to date, successfulinhibition or elimination of suckering has not been achieved throughthese approaches. Therefore, development of tobacco traits with limitedor no suckering would result in a reduction of the use of chemicalagents and would reduce costs and labor associated with tobaccoproduction.

SUMMARY

In one aspect, the present disclosure provides a modified tobacco plantcomprising no or reduced suckering compared to a control tobacco plantof the same variety when grown under comparable conditions.

In one aspect, the present disclosure provides a modified tobacco plant,where the modified tobacco plant exhibits: inhibited or eliminatedaxillary meristem growth; inhibited or eliminated axillary meristemmaintenance; or a combination thereof compared to a control tobaccoplant of the same variety when grown under comparable conditions.

In one aspect, the present disclosure provides a plant or seedcomprising a recombinant polynucleotide, where the recombinantpolynucleotide comprises a promoter that is functional in an L1 layer,an L2 layer, an L3 region, a rib zone, a central zone, a peripheralzone, or any combination thereof, which is operably linked to astructural nucleic acid molecule comprising a nucleic acid sequence,where the nucleic acid sequence encodes a polypeptide having at least70% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193,195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,and 255.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a promoter that is functional in an L1 layer, an L2layer, an L3 region, a rib zone, a central zone, a peripheral zone, or acombination thereof; and a heterologous and operably linked nucleic acidsequence, where the nucleic acid sequence encodes a non-coding RNA or apolypeptide.

In one aspect, the present disclosure provides a method of reducing oreliminating topping-induced suckering in a tobacco plant, where themethod comprises transforming a tobacco plant with a recombinant DNAconstruct comprising a promoter functional in an L1 layer, an L2 layer,an L3 region, a rib zone, a central zone, a peripheral zone, or acombination thereof.

In one aspect, the present disclosure provides a method comprisingtransforming a tobacco plant with a recombinant DNA construct comprisinga heterologous promoter that is functional in an L1 layer, an L2 layer,an L3 region, a rib zone, a central zone, a peripheral zone, or acombination thereof, and is operably linked to a polynucleotide that istranscribed into an RNA molecule that suppresses the level of anendogenous gene, and where the endogenous gene promotes or is requiredfor axillary meristem growth, axillary meristem maintenance, or both.

In one aspect, the present disclosure provides a method for producing atobacco plant comprising crossing at least one tobacco plant of a firsttobacco variety with at least one tobacco plant of a second tobaccovariety, where the at least one tobacco plant of the first tobaccovariety exhibits no or reduced topping-induced suckering compared to acontrol tobacco plant of the same variety grown under comparableconditions; and selecting for progeny tobacco plants that exhibit no orreduced topping-induced suckering compared to a control tobacco plant ofthe same cross grown under comparable conditions.

In one aspect, the present disclosure provides a tobacco plant, or partthereof, comprising a heterologous promoter operably linked to apolynucleotide encoding a polypeptide having at least 70% sequenceidentity to a polypeptide selected from the group consisting of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201,203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a heterologous promoter operably linked to apolynucleotide encoding a polypeptide having at least 70% sequenceidentity to a polypeptide selected from the group consisting of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201,203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, the present disclosure provides a method of growing amodified tobacco plant comprising planting a modified tobacco seedcomprising a heterologous promoter that is operably linked to apolynucleotide encoding a polypeptide having at least 70% sequenceidentity to a polypeptide selected from the group consisting of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201,203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255; and growingthe modified tobacco plant from the seed.

In one aspect, the present disclosure provides a method for controllingtopping-induced suckering in a plant comprising transforming the plantwith a recombinant DNA construct, where the recombinant DNA constructcomprises a promoter that is operably linked to a polynucleotideencoding a polypeptide having at least 70% sequence identity to apolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, the present disclosure provides a tobacco plant, or partthereof, comprising a heterologous promoter operably linked to apolynucleotide that encodes a non-coding RNA molecule, where thenon-coding RNA molecule is capable of binding to an RNA encoding apolypeptide having at least 70% sequence identity to a polypeptideselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 187,189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, and 255, and where the non-coding RNA moleculesuppresses the expression of the polypeptide.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a heterologous axillary meristem-specific promoteroperably linked to a polynucleotide that encodes a non-coding RNAmolecule, where the non-coding RNA molecule is capable of binding to anRNA encoding a polypeptide having at least 70% sequence identity to apolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78,161-185, 187, 189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, and 255, and where the non-coding RNAmolecule suppresses the expression of the polypeptide.

In one aspect, the present disclosure provides a method of growing amodified tobacco plant comprising planting a modified tobacco seedcomprising a recombinant DNA construct comprising a heterologouspromoter that is functional in an L1 layer, an L2 layer, an L3 region, arib zone, a central zone, a peripheral zone, or a combination thereof,and is operably linked to a polynucleotide that encodes a non-coding RNAmolecule, where the non-coding RNA molecule is capable of binding to anRNA encoding a polypeptide having at least 70% sequence identity to apolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78,161-185, 187, 189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, and 255, and where the non-coding RNAmolecule suppresses the expression of the polypeptide; and growing themodified tobacco plant from the seed.

In one aspect, the present disclosure provides a method for controllingtopping-induced suckering in a plant comprising transforming the plantwith a recombinant DNA construct, where the recombinant DNA constructcomprises a heterologous promoter that is functional in an L1 layer, anL2 layer, an L3 region, a rib zone, a central zone, a peripheral zone,or a combination thereof, and where the promoter is operably linked to apolynucleotide that encodes a non-coding RNA molecule, where thenon-coding RNA molecule is capable of binding to an RNA encoding apolypeptide having at least 70% sequence identity to a polypeptideselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 187,189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, and 255, and where the non-coding RNA moleculesuppresses the expression of the polypeptide.

In one aspect, the present disclosure provides a bacterial cellcomprising a recombinant DNA construct provided herein.

In one aspect, the present disclosure provides a plant genome comprisinga recombinant DNA construct provided herein.

In one aspect, the present disclosure provides a method formanufacturing a modified seed comprising introducing a recombinant DNAconstruct provided herein into a plant cell; screening a population ofplant cells for the recombinant DNA construct; selecting one or moreplant cells from the population, generating one or more modified plantsfrom the one or more plant cells; and collecting one or more modifiedseeds from the one or more modified plants.

In one aspect, the present disclosure provides a method of producing amodified tobacco plant to reduce or eliminate suckering, where themethod comprises introducing one or more mutations in one or moretobacco genome loci.

In one aspect, the present disclosure provides a method of producing amodified tobacco plant to reduce or eliminate suckering, where themethod comprises introducing one or more mutations in one or moretobacco genome loci, and where tobacco products are made from themodified tobacco plants.

In one aspect, the present disclosure provides a plant or seedcomprising a recombinant polynucleotide, where the recombinantpolynucleotide comprises a promoter that is functional in an L1 layer,an L2 layer, an L3 region, a rib zone, a central zone, a peripheralzone, or any combination thereof, which is operably linked to astructural nucleic acid molecule comprising a nucleic acid sequence,where the nucleic acid sequence encodes an auxin biosynthesis protein oran auxin transport protein.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a promoter that is functional in an L1 layer, an L2layer, an L3 region, a rib zone, a central zone, a peripheral zone, orany combination thereof; and a heterologous and operably linked nucleicacid sequence, where the nucleic acid sequence encodes an auxinbiosynthesis protein or an auxin transport protein.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a heterologous axillary meristem-specific promoteroperably linked to a polynucleotide that encodes an auxin biosynthesisprotein or an auxin transport protein.

In one aspect, the present disclosure provides a tobacco plant, or partthereof, comprising a heterologous promoter having at least 90% sequenceidentity to a polynucleotide selected from the group consisting of SEQID NOs: 113-118, 148-160, 204, and fragments thereof operably linked toa polynucleotide encoding an auxin biosynthesis protein or an auxintransport protein.

In one aspect, the present disclosure provides a method for controllingtopping-induced suckers in a plant comprising transforming said plantwith a recombinant DNA construct, where the recombinant DNA constructcomprises a promoter that is operably linked to a polynucleotideencoding an auxin biosynthesis protein or an auxin transport protein.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225-228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, and254 are nucleic acid sequences. SEQ ID NOs: 83-101 are RNAi constructs.SEQ ID NOs: 113-118, 148-160, and 204 are promoter or regulatory nucleicacid sequences. SEQ ID NOs: 119-122 are sequences used in TALENmutagenesis.

SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199,201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255 arepolypeptide sequences. Additional descriptions of the SEQ ID NOsprovided herein can be found in Table 1.

TABLE 1 Description of sequences SEQ Sequence NCBI Accession Number ofthe ID NO Type Sequence Description Top Hit of A BLAST Search 1 NucleicTranscription factor CYCLOIDEA-like XM_009609861.1 Acid 2 PeptideTranscription factor CYCLOIDEA-like 3 Nucleic Flower-specificgamma-thionin P32026.1 Acid 4 Peptide Flower-specific gamma-thionin 5Nucleic Polyphenoloxidase XP_006347083.1 Acid 6 PeptidePolyphenoloxidase 7 Nucleic UDP-glucose:glucosyltransferase BAG80546.1Acid 8 Peptide UDP-glucose:glucosyltransferase 9 Nucleic Tumor-relatedprotein BAA05479.1 Acid 10 Peptide Tumor-related protein 11 NucleicHypothetical protein CAN66732.1 Acid 12 Peptide Hypothetical protein 13Nucleic TCP1 protein-like gene FJ194953.1 Acid 14 Peptide TCP1protein-like gene 15 Nucleic Chlorophyllase-2 EYU43828.1 Acid 16 PeptideChlorophyllase-2 17 Nucleic AP2/ERF domain-containing transcriptionXP_006363442.1 Acid factor 18 Peptide AP2/ERF domain-containingtranscription factor 19 Nucleic Putative miraculin XP_006360306.1 Acid20 Peptide Putative miraculin 21 Nucleic Oleosin XP_004236249.1 Acid 22Peptide Oleosin 23 Nucleic ACC synthase XP_006356827.1 Acid 24 PeptideACC synthase 25 Nucleic LOB domain-containing protein 18-likeXP_007052037.1 Acid 26 Peptide LOB domain-containing protein 18-like 27Nucleic Vicilin-like antimicrobial peptides cupin XP_006363154.1 Acidsuper family 28 Peptide Vicilin-like antimicrobial peptides cupin superfamily 29 Nucleic Abscisic acid insensitive XP_006341248.1 Acid 30Peptide Abscisic acid insensitive 31 Nucleic Seipin-like XP_004237589.1Acid 32 Peptide Seipin-like 33 Nucleic Transcription factorCYCLOIDEA-like XM_009618194.1 Acid 34 Peptide Transcription factorCYCLOIDEA-like 35 Nucleic Transcription factor DICHOTOMA-likeXM_009593876.1 Acid 36 Peptide Transcription factor DICHOTOMA-like 37Nucleic Transcription factor CYCLOIDEA-like XM_009764845.1 Acid 38Peptide Transcription factor CYCLOIDEA-like 39 Nucleic RING-H2 fingerprotein ATL11-like XP_004251547.1 Acid 40 Peptide RING-H2 finger proteinATL11-like 41 Nucleic Homeobox-leucine zipper protein ATHB-XP_004232382.1 Acid 40-like 42 Peptide Homeobox-leucine zipper proteinATHB- 40- like 43 Nucleic Uncharacterized protein- LOC102586855XP_006357617.1 Acid isoform X1 44 Peptide Uncharacterized protein-LOC102586855 isoform X1 45 Nucleic Unknown CAN63006.1 Acid 46 PeptideUnknown 47 Nucleic MADS affecting flowering 5-like isoformXP_006366525.1 Acid X1/X2 48 Peptide MADS affecting flowering 5-likeisoform X1/X2 49 Nucleic Nuclear transcription factor Y subunitXP_006351227.1 Acid 50 Peptide Nuclear transcription factor Y subunit 51Nucleic Nuclear transcription factor Y subunit A-7- XP_006351229.1 Acidlike 52 Peptide Nuclear transcription factor Y subunit A-7- like 53Nucleic Transcription factor CYCLOIDEA-like XM_009767637.1 Acid 54Peptide Transcription factor CYCLOIDEA-like 55 Nucleic Arabidopsiscytokinin oxidase NM_129714.3 Acid 56 Peptide Arabidopsis cytokininoxidase 57 Nucleic Nicotiana tabacum cytokinin oxidase XM_009611148.1Acid 58 Peptide Nicotiana tabacum cytokinin oxidase 59 Nucleic Nicotianatabacum cytokinin oxidase XM_009632505.1 Acid 60 Peptide Nicotianatabacum cytokinin oxidase 61 Nucleic Nicotiana tabacum Isopentenyltransferase XM_009784416.1 Acid gene (IPT-g120126) 62 Peptide Nicotianatabacum Isopentenyl transferase gene (IPT-g120126) 63 Nucleic Nicotianatabacum WUSCHEL (WUS- XM_009589135.1 Acid g151887) 64 Peptide Nicotianatabacum WUSCHEL (WUS- g151887) 65 Nucleic Nicotiana tabacum WUSCHEL(WUS- XM_009793912.1 Acid g135280) 66 Peptide Nicotiana tabacum WUSCHEL(WUS- g135280) 67 Nucleic Arabidopsis thaliana CLAVATA3 (CLV3)NM_001124926.1 Acid 68 Peptide Arabidopsis thaliana CLAVATA3 (CLV3) 69Nucleic Nicotiana tabacum CLAVATA3 XM_009628563.1 Acid(Scaffold00010610) 70 Peptide Nicotiana tabacum CLAVATA3(Scaffold00010610) 71 Nucleic Nicotiana tabacum LATERAL XM_009619761.1Acid SUPPRESSOR (g56830) 72 Peptide Nicotiana tabacum LATERAL SUPPRESSOR(g56830) 73 Nucleic Nicotiana tabacum LATERAL XM_009766770.1 AcidSUPPRESSOR (scafflod0004261) 74 Peptide Nicotiana tabacum LATERALSUPPRESSOR (scafflod0004261) 75 Nucleic Nicotiana tabacum REGULATOR OFXM_009802273.1 Acid AXILLARY MERISTEMS (RAX- scaffold0000950) 76 PeptideNicotiana tabacum REGULATOR OF AXILLARY MERISTEMS (RAX- scaffold0000950)77 Nucleic Nicotiana tabacum REGULATOR OF XM_009602411.1 Acid AXILLARYMERISTEMS (RAX- scaffold00001904) 78 Peptide Nicotiana tabacum REGULATOROF AXILLARY MERISTEMS (RAX- scaffold00001904) 79 Nucleic Bacillusamyloliquefaciens extracellular CP009748.1 Acid ribonuclease (Barnase)80 Peptide Bacillus amyloliquefaciens extracellular ribonuclease(Barnase) 81 Nucleic Arabidopsis thaliana BRANCHED1 NM_001125184.1 Acid82 Peptide Arabidopsis thaliana BRANCHED1 83 Nucleic RNAi_1 (targetingSEQ ID NO: 1) Acid 84 Nucleic RNAi_2 (targeting SEQ ID NO: 3) Acid 85Nucleic RNAi_5 (targeting SEQ ID NO: 9) Acid 86 Nucleic RNAi_7(targeting SEQ ID NO: 13) Acid 87 Nucleic RNAi_8 (targeting SEQ ID NO:15) Acid 88 Nucleic RNAi_9 (targeting SEQ ID NO: 17) Acid 89 NucleicRNAi_10 (targeting SEQ ID NO: 19) Acid 90 Nucleic RNAi_12 (targeting SEQID NO: 21) Acid 91 Nucleic RNAi_14 (targeting SEQ ID NO: 25) Acid 92Nucleic RNAi_15 (targeting SEQ ID NO: 27) Acid 93 Nucleic RNAi_16(targeting SEQ ID NO: 29) Acid 94 Nucleic RNAi_17 (targeting SEQ ID NO:31) Acid 95 Nucleic RNAi_18 (targeting SEQ ID NO: 35) Acid 96 NucleicRNAi_26 (targeting SEQ ID NO: 49) Acid 97 Nucleic RNAi_61 (targeting SEQID NO: 61) Acid 98 Nucleic RNAi_63 and 65 (targeting SEQ ID NO: Acid 63and 65) 99 Nucleic RNAi_71 and 73 (targeting SEQ ID NO: Acid 71 and 73)100 Nucleic RNAi_75 and 77 (targeting SEQ ID NO: Acid 75 and 77) 101Nucleic RNAi_CET-26-6 (targeting SEQ ID NO: Acid 11, 49, 108, 109 and110) 102 Nucleic RNAi_45-2-7-TDNA-145337-RI Acid (targeting SEQ ID NO:39) 103 Nucleic RNAi_45-2-7-TDNA-348CDS-RI Acid (targeting SEQ ID NO:41) 104 Nucleic RNAi_45-2-7-TDNA-131180CDS-RI Acid (targeting SEQ ID NO:43) 105 Nucleic RNAi_45-2-7-TDNA-22266-RI (targeting Acid SEQ ID NO: 45)106 Nucleic RNAi_45-2-7-TDNA-53803/75660-RI Acid (targeting SEQ ID NO:49) 107 Nucleic RNAi_45-2-7-TDNA-21860-RI (targeting Acid SEQ ID NO: 47)108 Nucleic CEN-like protein 2 (CET2) g114109 AF145260.1 Acid 109Nucleic CEN-like protein 2 (CET2) g2420 XM_009596199.1 Acid 110 NucleicCEN-like protein 2 (CET2) XM_009787775.1 Acid Scaffold0003597 CDS 111Nucleic Transformation cassette Acid 112 Nucleic Agrobacteriumtransformation vector p45- Acid 2-7 113 Nucleic A promoter sequence of agene encoding Acid SEQ ID NO: 2 (Gene 1) 114 Nucleic A promoter sequenceof a gene encoding Acid SEQ ID NO: 8 (Gene 4) 115 Nucleic A promotersequence of a gene encoding Acid SEQ ID NO: 14 (Gene 7) 116 Nucleicpromoter of SEQ ID NO: 275 (Gene 11) Acid 117 Nucleic A promotersequence of a gene encoding Acid SEQ ID NO: 28 (Gene 15) 118 Nucleic Apromoter sequence of a gene encoding Acid SEQ ID NO: 4 (Gene 2) 119Nucleic Sequence for TALEN donor, which targets Acid a gene encoding SEQID NO: 1 120 Nucleic Sequence for TALEN binding sites, which Acidtargets a gene encoding SEQ ID NO: 1 121 Nucleic Sequence for TALEN,include promoter Acid NO: 118, NO: 113 which targets a gene encoding SEQID NO: 13 122 Nucleic Sequence for TALEN biding sites, which Acidtargets a gene encoding SEQ ID NO: 13 123 Nucleic Nicotiana tabacum T2Rnase XP_009794914.1 Acid 124 Nucleic Nicotiana tabacum T2 RnaseXP_009766067.1 Acid 125 Nucleic Nicotiana tabacum P1 RnaseXP_009597823.1 Acid 126 Nucleic Nicotiana tabacum Rnase XP_009775662.1Acid 127 Nucleic Nicotiana tabacum T2 Rnase XM_009794797.1 Acid 128Nucleic Nicotiana tabacum T2 Rnase XM_009627900.1 Acid 129 NucleicNicotiana tabacum T2 Rnase JQ041907.1 Acid 130 Nucleic Nicotiana tabacumT2 Rnase XM_009795594.1 Acid 131 Nucleic Nicotiana tabacum T2 RnaseXM_009795502.1 Acid 132 Nucleic Nicotiana tabacum 12 RnaseXM_009606804.1 Acid 133 Nucleic Nicotiana tabacum 12 RnaseXM_009794798.1 Acid 134 Nucleic Nicotiana tabacum 12 Rnase AB034638.1Acid 135 Nucleic Nicotiana tabacum 12 Rnase XM_009784762.1 Acid 136Nucleic Nicotiana tabacum 12 Rnase XM_009798107.1 Acid 137 Nucleic VPE14XM_009773063.1 Acid 138 Nucleic VPE15 XM_009594104.1 Acid 139 NucleicVPE16 XM_009784979.1 Acid 140 Nucleic VPE17 XM_009765910.1 Acid 141Nucleic VPE4 XM_009623321.1 Acid 142 Nucleic VPE6 XM_009764257.1 Acid143 Nucleic VPE7 AB075949.1 Acid 144 Nucleic Nicotiana tabacumProteinase XM_009801188.1 Acid 145 Nucleic Nicotiana tabacum ProteinaseXM_009792063.1 Acid 146 Nucleic Nicotiana tabacum ProteinaseXM_009779330.1 Acid 147 Nucleic Nicotiana tabacum ProteinaseXM_009764284.1 Acid 148 Nucleic Thionin 5′ upstream regulatory sequenceAcid 149 Nucleic Nicotiana tabacum Lateral Suppressor1 Acid (LAS1) 5′upstream regulatory sequence 150 Nucleic Nicotiana tabacum LAS1 3′downstream Acid regulatory sequence 151 Nucleic Nicotiana tabacum LAS25′ upstream Acid regulatory sequence 152 Nucleic Nicotiana tabacum LAS23′ downstream Acid regulatory sequence 153 Nucleic Nicotiana tabacumRegulator of Axillary Acid Meristems1 (RAX1) 5′ upstream regulatorysequence 154 Nucleic Nicotiana tabacum RAX1 3′ downstream Acidregulatory sequence 155 Nucleic Nicotiana tabacum RAX2 5′ upstream Acidregulatory sequence 156 Nucleic Nicotiana tabacum RAX2 3′ downstreamAcid regulatory sequence 157 Nucleic SEQ ID NO: 27 5′ upstreamregulatory Acid sequence 158 Nucleic SEQ ID NO: 27 3′ downstreamregulatory Acid sequence 159 Nucleic SEQ ID NO: 27 homolog 5′ upstreamAcid regulatory sequence 160 Nucleic SEQ ID NO: 27 homolog 3′ downstreamAcid regulatory sequence 161 Peptide Nicotiana tabacum T2 Rnase encodedby SEQ ID NO: 123 162 Peptide Nicotiana tabacum T2 Rnase encoded by SEQID NO: 124 163 Peptide Nicotiana tabacum P1 Rnase encoded by SEQ ID NO:125 164 Peptide Nicotiana tabacum Rnase encoded by SEQ ID NO: 126 165Peptide Nicotiana tabacum T2 Rnase encoded by SEQ ID NO: 127 166 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 128 167 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 129 168 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 130 169 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 131 170 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 132 171 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 133 172 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 134 173 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 135 174 PeptideNicotiana tabacum T2 Rnase encoded by SEQ ID NO: 136 175 Peptide VPE14encoded by SEQ ID NO: 137 176 Peptide VPE15 encoded by SEQ ID NO: 138177 Peptide VPE16 encoded by SEQ ID NO: 139 178 Peptide VPE17 encoded bySEQ ID NO: 140 179 Peptide VPE4 encoded by SEQ ID NO: 141 180 PeptideVPE6 encoded by SEQ ID NO: 142 181 Peptide VPE7 encoded by SEQ ID NO:143 182 Peptide Nicotiana tabacum Proteinase encoded by SEQ ID NO: 144183 Peptide Nicotiana tabacum Proteinase encoded by SEQ ID NO: 145 184Peptide Nicotiana tabacum Proteinase encoded by SEQ ID NO: 146 185Peptide Nicotiana tabacum Proteinase encoded by SEQ ID NO: 147 186Nucleic C12866 (Gene 11) XP_006467846.1 Acid 187 Peptide C12866 (Gene11) 188 Nucleic Nicotiana tabacum STM homolog AB004785 Acid (NTH15) 189Peptide Nicotiana tabacum STM homolog (NTH15) 190 Nucleic Nicotianatabacum (Grassy Tillers1) GT1 Acid homolog 191 Peptide Nicotiana tabacum(Grassy Tillers1) GT1 homolog 192 Nucleic Arabidopsis thaliana MoreAxillary AK316903.1 Acid Branching1 (MAX1) 193 Peptide Arabidopsisthaliana More Axillary Branching1 (MAX1) 194 Nucleic Arabidopsisthaliana MAX2 AAK97303.1 Acid 195 Peptide Arabidopsis thaliana MAX2 196Nucleic Nicotiana tabacum MAX1 homolog XM_009801023.1 Acid 197 PeptideNicotiana tabacum MAX1 homolog 198 Nucleic Nicotiana tabacum MAX2homolog XM_009625596.1 Acid 199 Peptide Nicotiana tabacum MAX2 homolog200 Nucleic Arabidopsis thaliana Lateral Suppressor BT026519.1 Acid(LAS) 201 Peptide Arabidopsis thaliana Lateral Suppressor (LAS) 202Nucleic Arabidopsis thaliana Regulator of Axillary AY519628.1 AcidMeristems (RAX) 203 Peptide Arabidopsis thaliana Regulator of AxillaryMeristems (RAX) 204 Nucleic Regulatory region of Solanum Acidlycopersicum homolog of SEQ ID NO: 28 205 Nucleic Solanum lycopersicumhomolog of SEQ ID HG975514.1 Acid NO: 28 206 Peptide Solanumlycopersicum homolog of SEQ ID NO: 28 207 Nucleic Arabidopsis thalianaALCATRAZ Acid 208 Peptide Arabidopsis thaliana ALCATRAZ 209 NucleicArabidopsis thaliana VND6 Acid 210 Peptide Arabidopsis thaliana VND6 211Nucleic Arabidopsis thaliana VND7 Acid 212 Peptide Arabidopsis thalianaVND7 213 Nucleic Solanum lycopersicum Adi3 Acid 214 Peptide Solanumlycopersicum Adi3 215 Nucleic Arabidopsis thaliana XCP1 Acid 216 PeptideArabidopsis thaliana XCP1 217 Nucleic Arabidopsis thaliana XCP2 Acid 218Peptide Arabidopsis thaliana XCP2 219 Nucleic Arabidopsis thalianaMetacaspase 2d Acid (ATMC4) 220 Peptide Arabidopsis thaliana Metacaspase2d (ATMC4) 221 Nucleic Arabidopsis thaliana disease resistance Acidprotein RPS5 222 Peptide Arabidopsis thaliana disease resistance proteinRPS5 223 Nucleic Nicotiana tabacum TMV resistance N gene Acid 224Peptide Nicotiana tabacum TMV resistance N gene 225 Nucleic Saccharumspp. mature miRNA159 Acid 226 Nucleic Nicotiana tabacum precursormiRNA159 Acid 227 Nucleic Nicotiana tabacum mature miRNA159 Acid 228Nucleic Nicotiana NAC089 Acid 229 Peptide Nicotiana NAC089 230 NucleicNicotiana BAG6 Acid 231 Peptide Nicotiana BAG6 232 Nucleic Nicotianamitogen-activated protein kinase Acid kinase 2 (NtMEK2) 233 PeptideNicotianamitogen-activated protein kinase kinase 2 (NtMEK2) 234 NucleicArabidopsis thaliana Flavin Acid monooxygenase (YUCCA1) 235 PeptideArabidopsis thaliana Flavin monooxygenase (YUCCA1) 236 NucleicArabidopsis thaliana Pin-formed1 (PIN1) Acid 237 Peptide Arabidopsisthaliana Pin-formed1 (PIN1) 238 Nucleic Arabidopsis thaliana TryptophanAcid aminotransferase1/Transport inhibitor response2 (TAA1/TIR2) 239Peptide Arabidopsis thaliana Tryptophan aminotransferase1/Transportinhibitor response2 (TAA1/TIR2) 240 Nucleic Arabidopsis thalianaAldehyde oxidase1 Acid (AAO1) 241 Peptide Arabidopsis thaliana Aldehydeoxidase1 (AAO1) 242 Nucleic Arabidopsis thaliana Indole-3-acetamide Acidhydrolase1 (AMI1) 243 Peptide Arabidopsis thaliana Indole-3-acetamidehydrolase1 (AMI1) 244 Nucleic Nicotiana Flavin monooxygenase Acid(NtYUCCA-like1) 245 Peptide Nicotiana Flavin monooxygenase(NtYUCCA-like1) 246 Nucleic Nicotiana Flavin monooxygenase Acid(NtYUCCA-like2) 247 Peptide Nicotiana Flavin monooxygenase(NtYUCCA-like2) 248 Nucleic Nicotiana Pin-formed1-like (NtPIN1-like)Acid 249 Peptide Nicotiana Pin-formed1-like (NtPIN1-like) 250 NucleicNicotiana Tryptophan Acid aminotransferase1/Transport inhibitorresponse2-like (NtTAA1/TIR2-like) 251 Peptide Nicotiana Tryptophanaminotransferase1/Transport inhibitor response2-like (NtTAA1/TIR2-like)252 Nucleic Nicotiana Aldehyde oxidase1-like Acid (NtAAO1-like) 253Peptide Nicotiana Aldehyde oxidase1-like (NtAAO1-like) 254 NucleicNicotiana Indole-3-acetamide hydrolase1- Acid like (NtAMI1-like) 255Peptide Nicotiana Indole-3-acetamide hydrolase1- like (NtAMI1-like) 256Peptide 6x Histidine tag

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map of binary vector p45-2-7 (SEQ ID NO: 112).

FIG. 2 shows photographs of control tobacco plants and modified tobaccoplants that overexpress SEQ ID NO: 11, which encodes a product thatpromotes sucker growth in tobacco. Plants are shown at the time oftopping (0 hour) and one week after topping. Modified plants exhibitincreased sucker growth compared to control plants.

FIG. 3 shows photographs of control tobacco plants and modified tobaccoplants that overexpress SEQ ID NO: 81, which encodes Arabidopsisthaliana BRANCHED1. FIG. 3A shows plants at the time of topping (0 hour)and one week after topping. Modified plants exhibit decreased suckergrowth compared to control plants. FIG. 3B shows that modified plantsoverexpressing SEQ ID NO: 81 exhibit stunted growth compared to controlplants.

FIG. 4 shows photographs of control tobacco plants and modified tobaccoplants that express SEQ ID NO: 83, an RNAi construct that targets SEQ IDNO: 1 for inhibition. Plants are shown at the time of topping (0 hour)and one week after topping. Modified plants exhibit enhanced suckeringcompared to control plants.

FIG. 5 shows the growth of axillary shoots in control tobacco plants andmodified tobacco plants that express SEQ ID NO: 83, an RNAi constructthat targets SEQ ID NO: 1 for inhibition. FIG. 5A shows photographs ofrepresentative control and modified tobacco plants, as well as all ofthe axillary shoots from one plant two weeks after topping. FIG. 5B is agraph displaying the total fresh weight of axillary shoots from controland modified plants two weeks after topping. Modified plants exhibitincreased axillary shoot mass compared to control plants.

FIG. 6 shows photographs of control tobacco plants and modified tobaccoplants that express SEQ ID NO: 86, an RNAi construct that targets SEQ IDNO: 13 for inhibition. Plants are shown at the time of topping (0 hour)and one week after topping. Modified plants exhibit enhanced suckeringcompared to control plants.

FIG. 7 is a graph displaying the total fresh weight of axillary shootsfrom control plants and modified tobacco plants that express SEQ ID NO:86, an RNAi construct that targets SEQ ID NO: 13 for inhibition, twoweeks after topping. Data from six independent modified tobacco linesare shown. Modified plants exhibit increased sucker mass compared tocontrol plants.

FIG. 8 shows photographs of modified tobacco plants that express SEQ IDNO: 95, an RNAi construct that targets SEQ ID NO: 35 for inhibition.Modified tobacco plants exhibit sucker outgrowth prior to topping.Arrows point to suckers.

FIG. 9 shows photographs of control tobacco plants and three independentlines of modified tobacco plants that express SEQ ID NO: 101, an RNAiconstruct that targets tobacco CENTRORADIALIS (SEQ ID NOs: 108-110).Photographs show the apex of a plant (top panel) or an entire plant(lower panel) one week after topping. Sucker growth is reduced inmodified plants.

FIG. 10 shows photographs of a control tobacco plant and a modifiedtobacco plant that expresses SEQ ID NO: 96, an RNAi construct thattargets SEQ ID NO: 49 for inhibition. Modified tobacco plants exhibitreduced sucker growth (arrows) compared to control tobacco plants.

FIG. 11 shows the expression pattern of Promoter P1 (SEQ ID NO: 113)fused to β-glucuronidase (GUS) in a tobacco shoot apical meristem (SAM)at the time of topping (0 hours) and in an axillary bud at 0 hours, 24hours after topping, and 72 hours after topping. Dark areas of GUSaccumulation demonstrate where Promoter P1 is active. Promoter P1 isfunctional in both shoot apical and axillary buds.

FIG. 12 shows the expression pattern of Promoter P11 (SEQ ID NO: 116)fused to β-glucuronidase (GUS) in a tobacco seedling; a tobacco seedlingshoot apical meristem (SAM); a mature SAM at the time of topping (0hours); and an axillary bud at the time of topping, 3 days aftertopping, 5 days after topping, and 7 days after topping. Dark areas ofGUS accumulation demonstrate where Promoter P11 is active. Promoter P11is weakly active in axillary buds prior to topping and has higheractivity in axillary buds 3 days after topping. Activity of Promoter P11decreases by 5 and 7 days after topping. Promoter P11 is also active inthe SAM prior to topping. No GUS staining is detected in seedlings.

FIG. 13 shows the expression pattern of Promoter P15 (SEQ ID NO: 117)fused to β-glucuronidase (GUS) in tobacco. Dark areas of GUSaccumulation demonstrate where Promoter P15 is active. FIG. 13A showsPromoter P15 activity in a tobacco seedling; a tobacco seedling shootapical meristem (SAM); a mature SAM at the time of topping (0 hours);and an axillary bud at the time of topping, 3 days after topping, 5 daysafter topping, 7 days after topping, and 15 days after topping. PromoterP15 is not active in seedlings. Promoter P15 is active at the base ofthe SAM, but it is not active in floral meristems. Promoter P15 exhibitsstrong activity in axillary buds prior to topping, and the activity ismaintained for at least 15 days after topping. FIG. 13B furtherdemonstrates the axillary bud specificity of Promoter P15. FIG. 13Bshows GUS expression driven by Promoter P15 at the time of topping (0hours), 7 days after topping, and 10 days after topping. At each timepoint, exemplary GUS staining of axillary buds from two independentmodified tobacco lines is shown. FIG. 13C displays GUS staining inmultiple axillary buds from individual plants at the time of topping (0hours), 24 hours after topping, and 72 hours after topping.

FIG. 14 shows microscopy photographs displaying the activity of axillarybud-specific promoters fused to green fluorescent protein (GFP). FIG.14A shows results of a Promoter P15 (SEQ ID NO: 117)::GFP fusion in ashoot apical meristem (SAM) and axillary bud (left panel) and in anaxillary bud (right panel). Promoter P15 activity is restricted toaxillary buds. FIG. 14B shows results of a Promoter P1 (SEQ ID NO:113)::GFP fusion in a shoot apical meristem (SAM) and axillary bud (leftpanel) and in an axillary bud (right panel).

FIG. 15 shows the expression pattern of an axillary bud Thionin promoter(pABTh, SEQ ID NO: 118) fused to β-glucuronidase (GUS) in tobacco apicaland axillary meristems at the time of topping (0 hours) and 30 hoursafter topping. Dark areas of GUS accumulation demonstrate where PromoterP15 is active.

FIG. 16 shows the locations of cis-regulatory elements in Promoter P1(SEQ ID NO: 113), Promoter P15 (SEQ ID NO: 117), and Promoter pABTh (SEQID NO: 118). +1 designates the transcriptional start site.

FIG. 17 shows photographs of control tobacco plants and modified tobaccoplants that express SEQ ID NO: 81, which encodes Arabidopsis thalianaBRANCHED1 and inhibits sucker growth, and SEQ ID NO: 101, an RNAiconstruct that targets tobacco CENTRORADIALIS and reduces sucker growth,driven by axillary bud-specific Promoter P15 (SEQ ID NO: 117). Plantsare shown 8 days after topping and 15 days after topping. Modifiedplants exhibit reduced sucker growth (arrows) compared to controlplants.

FIG. 18 shows photographs of control plants and a modified tobacco plantthat expresses SEQ ID NO: 59 (Nicotiana tabacum cytokinin oxidase 13)under the control of Promoter P15 (SEQ ID NO: 117) eight days aftertopping. Modified plants exhibit reduced sucker growth (arrows) comparedto control plants.

FIG. 19 shows photographs of control plants and two independent lines ofmodified tobacco plants that express SEQ ID NO: 79 (Barnase) under thecontrol of Promoter P15 (SEQ ID NO: 117). Modified plants exhibitreduced sucker growth (arrows) compared to control plants. No axillarymeristem primordia are observed before topping the modified plants.

FIG. 20 shows photographs of control plants and two independent lines ofmodified tobacco plants that express SEQ ID NO: 79 (Barnase) under thecontrol of Promoter P15 (SEQ ID NO: 117). Modified plants exhibitreduced sucker growth (arrows) compared to control plants one week aftertopping (FIG. 20A), two weeks after topping (FIG. 20B), and three weeksafter topping (FIG. 20C).

FIG. 21 shows photographs of tobacco leaves subjected toagroinfiltration as described in Example 15. A vector expressing SEQ IDNO: 79 (Barnase) is used as a positive control, and an vector lacking aninsert (empty vector) is used as a negative control. Genes of interestare examined ability to induce cellular death in a tobacco leafaccording to Example 15. For example, SEQ ID NO: 232 causes cellulardeath when expressed in a tobacco leaf.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. One skilled in the art will recognize many methods can be usedin the practice of the present disclosure. Indeed, the presentdisclosure is in no way limited to the methods and materials described.For purposes of the present disclosure, the following terms are definedbelow.

Any references cited herein, including, e.g., all patents, publishedpatent applications, and non-patent publications, are incorporated byreference in their entirety.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth.

As used herein, a tobacco plant can be from any plant from the Nicotianatabacum genus including, but not limited to Nicotiana tabacum tabacum;Nicotiana tabacum amplexicaulis PI 271989; Nicotiana tabacum benthamianaPI 555478; Nicotiana tabacum bigelovii PI 555485; Nicotiana tabacumdebneyi; Nicotiana tabacum excelsior PI 224063; Nicotiana tabacumglutinosa PI 555507; Nicotiana tabacum goodspeedii PI 241012; Nicotianatabacum gossei PI 230953; Nicotiana tabacum hesperis PI 271991;Nicotiana tabacum knightiana PI 555527; Nicotiana tabacum maritima PI555535; Nicotiana tabacum megalosiphon PI 555536; Nicotiana tabacumnudicaulis PI 555540; Nicotiana tabacum paniculata PI 555545; Nicotianatabacum plumbaginifolia PI 555548; Nicotiana tabacum repanda PI 555552;Nicotiana tabacum rustica; Nicotiana tabacum suaveolens PI 230960;Nicotiana tabacum sylvestris PI 555569; Nicotiana tabacum tomentosa PI266379; Nicotiana tabacum tomentosiformis; and Nicotiana tabacumtrigonophylla PI 555572.

In one aspect, this disclosure provides methods and compositions relatedto modified tobacco plants, seeds, plant components, plant cells, andproducts made from modified tobacco plants, seeds, plant parts, andplant cells. In one aspect, a modified seed provided herein gives riseto a modified plant provided herein. In one aspect, a modified plant,seed, plant component, plant cell, or plant genome provided hereincomprises a recombinant DNA construct provided herein. In anotheraspect, cured tobacco material or tobacco products provided hereincomprise modified tobacco plants, plant components, plant cells, orplant genomes provided herein.

As used herein, “modified” refers to plants, seeds, plant components,plant cells, and plant genomes that have been subjected to mutagenesis,genome editing, genetic transformation, or a combination thereof.

In one aspect, modified tobacco plants provided herein exhibit no orreduced suckering compared to control tobacco plants of the same varietywhen grown under comparable conditions. In one aspect, modified tobaccoplants provided herein exhibit no or reduced topping-induced suckeringcompared to control tobacco plants of the same variety when grown undercomparable conditions. Also provided herein are methods of producingmodified tobacco plants that exhibit no or reduced suckering compared tocontrol tobacco plants of the same variety when grown under comparableconditions. In one aspect, methods provided herein produce modifiedtobacco plants that exhibit no or reduced topping-induced suckeringcompared to control tobacco plants of the same variety when grown undercomparable conditions.

As used herein, “cisgenesis” or “cisgenic” refers to geneticmodification of a plant, plant cell, or plant genome in which allcomponents (e.g., promoter, donor nucleic acid, selection gene) haveonly plant origins (i.e., no non-plant origin components are used). Inone aspect, a modified plant, plant cell, or plant genome providedherein is cisgenic. Cisgenic plants, plant cells, and plant genomesprovided herein can lead to ready-to-use tobacco lines. In anotheraspect, a modified tobacco plant provided herein comprises nonon-tobacco genetic material or sequences.

As used herein, “suckering” refers to the development and/or growth ofaxillary (or lateral) buds (“suckers”) from axillary meristems that growbetween a leaf and the stalk. An axillary bud is an embryonic shoot thatcomprises an axillary meristem, surrounding leaf tissue, and surroundingstem tissue. In one aspect, suckering is induced by topping a plant.

As used herein, “topping” refers to the removal of the stalk apex,including the SAM, flowers, and up to several adjacent leaves, when aplant is near maturity. Topping a tobacco plant results in the loss ofapical dominance. Prior to topping, suckering is largely kept dormant byhormonal signals emanating from the SAM; topping removes the hormonalsignals and can allow the outgrowth of suckers (“topping-inducedsuckering”). Provided suckering is sufficiently controlled, toppingincreases yield, increases value-per-acre, and results in desirablemodifications to physical and chemical properties of tobacco leaves.

As used herein, “comparable growth conditions” refers to similarenvironmental conditions and/or agronomic practices for growing andmaking meaningful comparisons between two or more plant genotypes sothat neither environmental conditions nor agronomic practices wouldcontribute to, or explain, any differences observed between the two ormore plant genotypes. Environmental conditions include, for example,light, temperature, water, humidity, and nutrition (e.g., nitrogen andphosphorus). Agronomic practices include, for example, seeding,clipping, undercutting, transplanting, topping, and suckering. SeeChapters 4B and 4C of Tobacco, Production, Chemistry and Technology,Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70-103.

As used herein, “reduced topping-induced suckering” refers to areduction in the number of suckers; a reduction in the size of suckers(e.g., biomass), and/or a reduction of the impact suckers have onagronomic performance (e.g., yield, quality and overall productivity ofthe plant) compared to a control plant when grown under comparableconditions. As used herein, a “reduction” in the number of suckers, thesize of suckers, and/or the impact suckers have on agronomic performancerefers to a statistically significant reduction. As used herein,“statistically significant” refers to a p-value of less than 0.05, ap-value of less than 0.025, a p-value of less than 0.01, or a p-value ofless than 0.001 when using an appropriate measure of statisticalsignificance (e.g., a one-tailed two sample t-test).

The present disclosure provides modified tobacco plants with desirableor enhanced properties, e.g., inhibited or reduced sucker growth priorto or after topping. In one aspect, a modified plant provided hereincomprises fewer total suckers, smaller suckers, or both compared to acontrol plant lacking such modification when grown under comparableconditions. In one aspect, smaller suckers of a modified plant providedherein comprise reduced mass, reduced length, reduced diameter, or acombination thereof compared to suckers of a control plant grown undercomparable conditions. The diameter of a sucker is measured at the baseof the sucker where it adjoins the main stem of the plant.

In one aspect, the mass of suckers of a modified tobacco plant providedherein is at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%reduced compared to the mass of suckers of an unmodified control tobaccoplant grown under comparable conditions. In one aspect, the mass ofsuckers of a modified tobacco plant provided herein is reduced by1%-25%, 1%-50%, 1%-75%, 1%-100%, 5%-25%, 5%-50%, 5%-75%, 5%-100%,10%-25%, 10%-50%, 10%-75%, 10%-100%, 25%-50%, 25%-75%, 25%-100%,50%-75%, or 50%-100% as compared to the mass of suckers of an unmodifiedcontrol tobacco plant grown under comparable conditions. In anotheraspect, the length of suckers of a modified tobacco plant providedherein is at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%reduced compared to the length of suckers of an unmodified controltobacco plant grown under comparable conditions. In one aspect, thelength of suckers of a modified tobacco plant provided herein is reducedby 1%-25%, 1%-50%, 1%-75%, 1%-100%, 5%-25%, 5%-50%, 5%-75%, 5%-100%,10%-25%, 10%-50%, 10%-75%, 10%-100%, 25%-50%, 25%-75%, 25%-100%,50%-75%, or 50%-100% as compared to the length of suckers of anunmodified control tobacco plant grown under comparable conditions. Inone aspect, the diameter of suckers of a modified tobacco plant providedherein is at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%reduced compared to the diameter of suckers of an unmodified controltobacco plant grown under comparable conditions. In one aspect, thediameter of suckers of a modified tobacco plant provided herein isreduced by 1%-25%, 1%-50%, 1%-75%, 1%-100%, 5%-25%, 5%-50%, 5%-75%,5%-100%, 10%-25%, 10%-50%, 10%-75%, 10%-100%, 25%-50%, 25%-75%,25%-100%, 50%-75%, or 50%-100% as compared to the diameter of suckers ofan unmodified control tobacco plant grown under comparable conditions.

In another aspect, a modified tobacco plant provided herein comprises atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20,at least 30, at least 40, at least 50, or at least 60 fewer totalsuckers compared to an unmodified control tobacco plant grown undercomparable conditions. In another aspect, a modified tobacco plantprovided herein comprises at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% fewer suckers compared to an unmodified control tobacco plantgrown under comparable conditions. In one aspect, the number of suckersof a modified tobacco plant provided herein is reduced by 1%-25%,1%-50%, 1%-75%, 1%-100%, 5%-25%, 5%-50%, 5%-75%, 5%-100%, 10%-25%,10%-50%, 10%-75%, 10%-100%, 25%-50%, 25%-75%, 25%-100%, 50%-75%, or50%-100% as compared to the number of suckers of an unmodified controltobacco plant grown under comparable conditions.

Shoot apical and axillary meristems have two main functions: to maintainthemselves as a group of pluripotent cells, and to generate lateralabove-ground organs of the plant (e.g., stems, leaves, flowers). If ameristem fails to maintain itself, for any reason, it will eventuallyexhaust its pluripotent cells and cease giving rise to additionalorgans. In one aspect, a modified tobacco plant provided herein exhibitsinhibited or eliminated axillary meristem growth; inhibited oreliminated axillary meristem maintenance; or a combination thereofcompared to a control tobacco plant of the same variety when grown undercomparable conditions.

In one aspect, a modified tobacco plant provided herein has similar orhigher leaf yield compared to a control tobacco plant when grown undercomparable conditions. In an aspect, leaf yield is selected from thegroup consisting of fresh yield, dry yield, and cured yield. In oneaspect, a modified tobacco plant provided herein produces a leaf yieldmass within about 50%, within about 45%, within about 40%, within about35%, within about 30%, within about 25%, within about 20%, within about15%, within about 10%, within about 5%, within about 4%, within about3%, within about 2%, within about 1%, or within about 0.5% compared to acontrol tobacco plant when grown under comparable conditions. In anotheraspect, a modified tobacco plant provided herein produces a leaf yieldmass at least 0.25%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or more than 100% higher compared to acontrol tobacco plant when grown under comparable conditions. In anotheraspect, a modified tobacco plant provided herein produces a leaf yieldmass 0.25%-100%, 0.5%-100%, 1%-100%, 2.5%-100%, 5%-100%, 10%-100%,15%-100%, 20%-100%, 25%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%,70%-100%, 80%-100%, 90%-100%, 100%-200%, 100%-175%, 100%-150%,100%-125%, 0.25%-50%, 0.5%-50%, 1%-50%, 2.5%-50%, 5%-50%, 10%-50%,15%-50%, 20%-50%, 25%-50%, 30%-50%, 40%-50%, 50%-200%, 50%-175%,50%-150%, 50%-125%, 0.25%-25%, 0.5%-25%, 1%-25%, 2.5%-25%, 5%-25%,10%-25%, 15%-25%, 20%-25%, 25%-200%, 25%-175%, 25%-150%, or 25%-125%higher compared to a control tobacco plant when grown under comparableconditions. In an aspect, a modified tobacco plant provided hereinproduces a number of leaves within 75%, within 60%, within 50%, within45%, within 40%, within 35%, within 30%, within 25%, within 20%, within15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%,or within 0.5% the number of leaves produced by an unmodified controltobacco plant grown under comparable conditions.

In one aspect, a modified tobacco plant provided herein has a similar orcomparable plant height compared to a control tobacco plant when grownunder comparable conditions. In one aspect, a modified tobacco plantprovided herein comprises a height within about 50%, within about 45%,within about 40%, within about 35%, within about 30%, within about 25%,within about 20%, within about 15%, within about 10%, within about 5%,within about 4%, within about 3%, within about 2%, within about 1%, orwithin about 0.5% compared to a control tobacco plants when grown undercomparable conditions. In another aspect, a modified tobacco plantprovided herein comprises a height 0.25%, 0.5%, 1%, 2.5%, 5%, 10%, 15%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%taller compared to a control tobacco plant when grown under comparableconditions. In another aspect, a modified tobacco plant comprises aheight 0.25%-100%, 0.5%-100%, 1%-100%, 2.5%-100%, 5%-100%, 10%-100%,15%-100%, 20%-100%, 25%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%,70%-100%, 80%-100%, 90%-100%, 100%-200%, 100%-175%, 100%-150%,100%-125%, 0.25%-50%, 0.5%-50%, 1%-50%, 2.5%-50%, 5%-50%, 10%-50%,15%-50%, 20%-50%, 25%-50%, 30%-50%, 40%-50%, 50%-200%, 50%-175%,50%-150%, 50%-125%, 0.25%-25%, 0.5%-25%, 1%-25%, 2.5%-25%, 5%-25%,10%-25%, 15%-25%, 20%-25%, 25%-200%, 25%-175%, 25%-150%, or 25%-125%taller compared to a control tobacco plant when grown under comparableconditions.

In one aspect, a modified tobacco plant provided herein produces leavesthat have a similar or higher USDA grade index value compared to acontrol tobacco plant when grown under comparable conditions. In oneaspect, a modified tobacco plant provided herein produces leaves with aUSDA grade index value within about 50%, within about 45%, within about40%, within about 35%, within about 30%, within about 25%, within about20%, within about 15%, within about 10%, within about 5%, within about4%, within about 3%, within about 2%, within about 1%, or within about0.5% compared to a control tobacco plant when grown under comparableconditions. In one aspect, a modified tobacco plant provided herein iscapable of producing leaves having a USDA grade index value of 55 ormore, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 ormore, 90 or more, or 95 or more. In one aspect, a modified tobacco plantprovided herein produces leaves with a USDA grade index value at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50units higher compared to a control tobacco plant when grown undercomparable conditions. In one aspect, a modified tobacco plant providedherein produces leaves with a USDA grade index value 1-50, 1-45, 1-40,1-35, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20,1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8,1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-45, 2-40, 2-35, 2-30, 2-29, 2-28,2-27, 2-26, 2-25, 2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16,2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,3-50, 3-45, 3-40, 3-35, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23,3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11,3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-50, 4-45, 4-40, 4-35, 4-30, 4-29,4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17,4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-50,5-45, 5-40, 5-35, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22,5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10,5-9, 5-8, 5-7, 5-6, 10-50, 10-40, 10-30, 10-20, 20-50, 20-30, 20-40, or20-30 units higher compared to a control tobacco plant when grown undercomparable conditions.

Tobacco grades are evaluated based on factors including, but not limitedto, the leaf stalk position, leaf size, leaf color, leaf uniformity andintegrity, ripeness, texture, elasticity, sheen (related with theintensity and the depth of coloration of the leaf as well as the shine),hygroscopicity (the faculty of the tobacco leaves to absorb and toretain the ambient moisture), and green nuance or cast. Leaf grade canbe determined, for example, using an Official Standard Grade publishedby the Agricultural Marketing Service of the US Department ofAgriculture (7 U.S.C. §511). See, e.g., Official Standard Grades forBurley Tobacco (U.S. Type 31 and Foreign Type 93), effective Nov. 5,1990 (55 F.R. 40645); Official Standard Grades for Flue-Cured Tobacco(U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective Mar. 27, 1989(54 F.R. 7925); Official Standard Grades for Pennsylvania SeedleafTobacco (U.S. Type 41), effective Jan. 8, 1965 (29 F.R. 16854); OfficialStandard Grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44),effective Dec. 8, 1963 (28 F.R. 11719 and 28 F.R. 11926); OfficialStandard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and55), effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Gradesfor Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effectiveNov. 20, 1969 (34 F.R. 17061); Official Standard Grades for Georgia andFlorida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), EffectiveApril 1971. A USDA grade index value can be determined according to anindustry accepted grade index. See, e.g., Bowman et al, Tobacco Science,32:39-40(1988); Legacy Tobacco Document Library (Bates Document#523267826-523267833, Jul. 1, 1988, Memorandum on the Proposed BurleyTobacco Grade Index); and Miller et al., 1990, Tobacco Intern.,192:55-57 (all foregoing references are incorporated by inference intheir entirety). Alternatively, leaf grade can be determined viahyper-spectral imaging. See e.g., WO 2011/027315 (published on Mar. 10,2011, and incorporated by inference in its entirety).

In one aspect, a modified plant provided herein requires reducedmanagement for controlling suckering compared to a control plant whengrown under comparable conditions. As used herein, “management” refersto manually removing suckers, application of chemicals (e.g., maleichydrazide, flumetralin) to inhibit or remove suckers, or both. In oneaspect, a modified plant provided herein requires reduced frequency ofmanual sucker removal, reduced frequency of chemical application,reduced quantities of chemical application, or a combination thereof,compared to a control plant grown under comparable conditions. See, forexample, Fisher et al. “Topping, Managing Suckers, and Using Ethephon,”pages 96-117 In: 2016 Flue-Cured Tobacco Information, North CarolinaState University, which is herein incorporated by reference in itsentirety. In one aspect, a modified plant provided herein requiresmanual removal of suckers 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, or 95% as frequently as a control plant when grown undercomparable conditions. In one aspect, a modified plant provided hereinrequires manual removal of suckers less than 10%, less than 20%, lessthan 30%, less than 40%, less than 50%, less than 60%, less than 70%,less than 75%, less than 80%, less than 85%, less than 90%, or less than95% as frequently as a control plant when grown under comparableconditions. In one aspect, a modified plant provided herein requiresmanual removal of suckers 10%-95%, 20%-95%, 30%-95%, 40%-95%, 50%-95%,60%-95%, 70%-95%, 80%-95%, 85%-95%, 90%-95%, 10%-50%, 20%-50%, 30%-50%,40%-50%, 10%-20%, 10%-30%, 10%-40%, 10%-50%, 10%-60%, 10%-70%, 10%-80%,10%-85%, or 10%-90% as frequently as a control plant when grown undercomparable conditions. In one aspect, a modified plant provided hereinrequires chemical application to control suckering 10%, 20%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% as frequently as a controlplant when grown under comparable conditions. In one aspect, a modifiedplant provided herein requires chemical application to control suckeringless than 10%, less than 20%, less than 30%, less than 40%, less than50%, less than 60%, less than 70%, less than 75%, less than 80%, lessthan 85%, less than 90%, or less than 95% as frequently as a controlplant when grown under comparable conditions. In one aspect, a modifiedplant provided herein requires chemical application to control suckering10%-95%, 20%-95%, 30%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95%, 80%-95%,85%-95%, 90%-95%, 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-20%, 10%-30%,10%-40%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-85%, or 10%-90% asfrequently as a control plant when grown under comparable conditions. Inone aspect, a modified plant provided herein requires a chemical sprayvolume of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%of the volume used to control suckering of a control plant when grownunder comparable conditions. In one aspect, a modified plant providedherein requires a chemical spray volume of less than 10%, less than 20%,less than 30%, less than 40%, less than 50%, less than 60%, less than70%, less than 75%, less than 80%, less than 85%, less than 90%, or lessthan 95% of the volume used to control suckering of a control plant whengrown under comparable conditions. In one aspect, a modified plantprovided herein requires a chemical spray volume 10%-95%, 20%-95%,30%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95%, 80%-95%, 85%-95%, 90%-95%,10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-20%, 10%-30%, 10%-40%, 10%-50%,10%-60%, 10%-70%, 10%-80%, 10%-85%, 10%-90% less than a control plantwhen grown under comparable conditions.

Unless specified otherwise, measurements of sucker length, sucker mass,number of suckers, leaf yield, or leaf grade index values mentionedherein for a tobacco plant, variety, cultivar, or line refer to averagemeasurements, including, for example, an average of multiple leaves(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 or more leaves) of a single plant or an average measurement from apopulation of tobacco plants from a single variety, cultivar, or line. Apopulation of tobacco plants or a collection of tobacco leaves fordetermining an average measurement (e.g., fresh weight or leaf grading)can be of any size, for example, 5, 10, 15, 20, 25, 30, 35, 40, or 50.Industry-accepted standard protocols are followed for determiningaverage measurements or grade index values.

In one aspect, a modified plant or leaf has a similar leaf chemistryprofile compared to a control plant when grown under comparableconditions. Without being limiting, a leaf chemistry profile cancomprise the amount of alkaloids (e.g., nicotine, nornicotine,anabasine, anatabine), malic acid, and reducing sugars (e.g., dextrose),or a combination thereof in a tobacco plant or tobacco leaf. In oneaspect, a modified plant or leaf provided herein comprises a totalalkaloids level within about 50%, within about 45%, within about 40%,within about 35%, within about 30%, within about 25%, within about 20%,within about 15%, within about 10%, within about 5%, within about 4%,within about 3%, within about 2%, within about 1%, or within about 0.5%of the total alkaloids level of a control plant when grown undercomparable conditions. In one aspect, a modified plant or leaf providedherein comprises a nicotine level within about 50%, within about 45%,within about 40%, within about 35%, within about 30%, within about 25%,within about 20%, within about 15%, within about 10%, within about 5%,within about 4%, within about 3%, within about 2%, within about 1%, orwithin about 0.5% of the nicotine level of a control plant when grownunder comparable conditions. In one aspect, a modified plant or leafprovided herein comprises a nornicotine level within about 50%, withinabout 45%, within about 40%, within about 35%, within about 30%, withinabout 25%, within about 20%, within about 15%, within about 10%, withinabout 5%, within about 4%, within about 3%, within about 2%, withinabout 1%, or within about 0.5% of the nornicotine level of a controlplant when grown under comparable conditions. In one aspect, a modifiedplant or leaf provided herein comprises an anabasine level within about50%, within about 45%, within about 40%, within about 35%, within about30%, within about 25%, within about 20%, within about 15%, within about10%, within about 5%, within about 4%, within about 3%, within about 2%,within about 1%, or within about 0.5% of the anabasine level of acontrol plant when grown under comparable conditions. In one aspect, amodified plant or leaf provided herein comprises an anatabine levelwithin about 50%, within about 45%, within about 40%, within about 35%,within about 30%, within about 25%, within about 20%, within about 15%,within about 10%, within about 5%, within about 4%, within about 3%,within about 2%, within about 1%, or within about 0.5% of the anatabinelevel of a control plant when grown under comparable conditions. In oneaspect, a modified plant or leaf provided herein comprises a malic acidlevel within about 50%, within about 45%, within about 40%, within about35%, within about 30%, within about 25%, within about 20%, within about15%, within about 10%, within about 5%, within about 4%, within about3%, within about 2%, within about 1%, or within about 0.5% of the malicacid level of a control plant when grown under comparable conditions. Inone aspect, a modified plant or leaf provided herein comprises areducing sugars level within about 50%, within about 45%, within about40%, within about 35%, within about 30%, within about 25%, within about20%, within about 15%, within about 10%, within about 5%, within about4%, within about 3%, within about 2%, within about 1%, or within about0.5% of the reducing sugars level of a control plant when grown undercomparable conditions. In one aspect, a modified plant or leaf providedherein comprises a dextrose level within about 50%, within about 45%,within about 40%, within about 35%, within about 30%, within about 25%,within about 20%, within about 15%, within about 10%, within about 5%,within about 4%, within about 3%, within about 2%, within about 1%, orwithin about 0.5% of the dextrose level of a control plant when grownunder comparable conditions.

In one aspect, a modified plant or leaf provided herein comprises no orreduced suckers compared to a control plant and further comprises anicotine level at least 50%, at least 60%, at least 70%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or at least 99.9% lower than the nicotinelevel of a control plant when grown under comparable conditions.

In one aspect, a plant component provided herein includes, but is notlimited to, a leaf, a stem, a root, a seed, a flower, pollen, an anther,an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, apod, an embryo, endosperm, an explant, a callus, a tissue culture, ashoot, a cell, and a protoplast. In further aspects, this disclosureprovides tobacco plant cells, tissues, and organs that are notreproductive material and do not mediate the natural reproduction of theplant. In another aspect, this disclosure also provides tobacco plantcells, tissues, and organs that are reproductive material and mediatethe natural reproduction of the plant. In another aspect, thisdisclosure provides tobacco plant cells, tissues, and organs that cannotmaintain themselves via photosynthesis. In another aspect, thisdisclosure provides somatic tobacco plant cells. Somatic cells, contraryto germline cells, do not mediate plant reproduction.

Provided cells, tissues and organs can be from seed, fruit, leaf,cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot, stem,pod, flower, inflorescence, stalk, pedicel, style, stigma, receptacle,petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem,and vascular tissue. In another aspect, this disclosure provides atobacco plant chloroplast. In a further aspect, this disclosure providesan epidermal cell, a stomata cell, a leaf hair (trichome), a root hair,or a storage root. In another aspect, this disclosure provides a tobaccoprotoplast.

Skilled artisans understand that tobacco plants naturally reproduce viaseeds, not via asexual reproduction or vegetative propagation. In oneaspect, this disclosure provides tobacco endosperm. In another aspect,this disclosure provides a tobacco endosperm cell. In a further aspect,this disclosure provides a male or female sterile tobacco plant, whichcannot reproduce without human intervention.

In one aspect, a modified plant, seed, plant part, or plant cellprovided herein comprises one or more non-naturally occurring mutations.In one aspect, a mutation provided herein suppresses suckering in aplant. In another aspect, a mutation provided herein suppressestopping-induced suckering in a plant. In still another aspect, amutation provided herein suppresses suckering in a plant prior totopping. Types of mutations provided herein include, for example,substitutions (point mutations), deletions, insertions, duplications,and inversions. Such mutations are desirably present in the codingregion of a gene; however, mutations in a promoter or other regulatoryregion, an intron, an intron-exon boundary, or an untranslated region ofa gene may also be desirable.

In one aspect, methods provided herein are capable of producing atobacco plant with reduced suckering using mutagenesis. Mutagenesismethods include, without limitation, chemical mutagenesis, for example,treatment of seeds with ethyl methylsulfate (EMS) (Hildering andVerkerk, In, The use of induced mutations in plant breeding. PergamonPress, pp. 317-320, 1965); or UV-irradiation, X-rays, electron beams,ion beams (e.g., carbon ion beam, helium ion beam, neon ion beam), andfast neutron irradiation (see, for example, Verkerk, Neth. J. Agric.Sci. 19:197-203, 1971; Poehlman, Breeding Field Crops, Van NostrandReinhold, New York (3.sup.rd ed.), 1987; and Tanaka, J. Radiat. Res.51:223-233, 2010); transposon tagging (Fedoroff et al., 1984; U.S. Pat.No. 4,732,856 and U.S. Pat. No. 5,013,658); and T-DNA insertionmethodologies (Hoekema et al., 1983; U.S. Pat. No. 5,149,645).EMS-induced mutagenesis consists of chemically inducing random pointmutations over the length of a genome. Fast neutron mutagenesis consistsof exposing seeds to neutron bombardment which causes large deletionsthrough double stranded DNA breakage. Transposon tagging comprisesinserting a transposon within an endogenous gene to reduce or eliminateexpression of the gene.

In addition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the present disclosure. See, McCallumet al. (2000) Nat. Biotechnol. 18:455-457. Mutations that impact geneexpression or that interfere with the function of genes provided hereincan be determined using methods that are well known in the art.Insertional mutations in gene exons usually result in null-mutants.Mutations in conserved residues can be particularly effective ininhibiting the function of a protein.

The screening and selection of mutagenized tobacco plants can be throughany methodologies known to those having ordinary skill in the art.Examples of screening and selection methodologies include, but are notlimited to, Southern analysis, PCR amplification for detection of apolynucleotide, Northern blots, RNase protection, primer-extension,RT-PCR amplification for detecting RNA transcripts, Sanger sequencing,Next Generation sequencing technologies (e.g., Illumina, PacBio, IonTorrent, 454) enzymatic assays for detecting enzyme or ribozyme activityof polypeptides and polynucleotides, and protein gel electrophoresis,Western blots, immunoprecipitation, and enzyme-linked immunoassays todetect polypeptides. Other techniques such as in situ hybridization,enzyme staining, and immunostaining also can be used to detect thepresence or expression of polypeptides and/or polynucleotides. Methodsfor performing all of the referenced techniques are known.

In one aspect, a polynucleotide provided herein comprises at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, or more than 10 mutations compared toa naturally existing polynucleotide. In another aspect, a mutationprovided herein is positioned within a polynucleotide selected from thegroup consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240, 242, 244, 246,248, 250, 252, and 254. In one aspect, a mutation provided herein ispositioned within a polynucleotide encoding a polypeptide selected fromthe group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189,191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218,220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, and 255.

In one aspect, a plant genome provided herein is mutated (edited) by anuclease selected from the group consisting of a meganuclease, azinc-finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), a CRISPR/Cas9 nuclease, or a CRISPR/Cpf1 nuclease. Inanother aspect, a plant genome provided herein is mutated by aCRISPR/CasX or a CRISPR/CasY nuclease. As used herein, “editing” or“genome editing” refers to targeted mutagenesis of at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least8, at least 9, or at least 10 nucleotides of an endogenous plant genomenucleic acid sequence, or removal or replacement of an endogenous plantgenome nucleic acid sequence. In one aspect, an edited nucleic acidsequence provided herein has at least 99.9%, at least 99.5%, at least99%, at least 98%, at least 97%, at least 96%, at least 95%, at least94%, at least 93%, at least 92%, at least 91%, at least 90%, at least85%, at least 80%, or at least 75% sequence identity with an endogenousnucleic acid sequence. In one aspect, an edited nucleic acid sequenceprovided herein has at least 99.9%, at least 99.5%, at least 99%, atleast 98%, at least 97%, at least 96%, at least 95%, at least 94%, atleast 93%, at least 92%, at least 91%, at least 90%, at least 85%, atleast 80%, or at least 75% sequence identity with a polynucleotideselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, and fragments thereof. In anotheraspect, an edited nucleic acid sequence provided herein has at least99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, atleast 96%, at least 95%, at least 94%, at least 93%, at least 92%, atleast 91%, at least 90%, at least 85%, at least 80%, or at least 75%sequence identity with a polynucleotide encoding a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255.

In one aspect, a nuclease provided herein is used to edit a plantgenomic locus encoding a sequence having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to apolynucleotide selected from the group consisting of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83-160, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, and fragments thereof.

In another aspect, a nuclease provided herein is used to edit a plantgenomic locus encoding a polypeptide having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to apolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, and 255. In another aspect, anuclease provided herein is used to edit a plant genome locus encoding apolypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence similarity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, and CRISPR/Cpf1 induce adouble-strand DNA break at a target site of a genomic sequence that isthen repaired by the natural processes of homologous recombination (HR)or non-homologous end-joining (NHEJ). Sequence modifications then occurat the cleaved sites, which can include deletions or insertions thatresult in gene disruption in the case of NHEJ, or integration of donornucleic acid sequences by HR. In one aspect, a methods provided hereincomprises editing a plant genome with a nuclease provided herein tomutate at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, or more than10 nucleotides in the plant genome via HR with a donor polynucleotide.In one aspect, a mutation provided herein is caused by genome editingusing a nuclease. In another aspect, a mutation provided herein iscaused by non-homologous end-joining or homologous recombination.

Meganucleases, which are commonly identified in microbes, are uniqueenzymes with high activity and long recognition sequences (>14 bp)resulting in site-specific digestion of target DNA. Engineered versionsof naturally occurring meganucleases typically have extended DNArecognition sequences (for example, 14 to 40 bp).

In one aspect, a meganuclease provided herein edits a polynucleotidehaving at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to a polynucleotide selected from thegroup consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240, 242, 244, 246,248, 250, 252, 254, and fragments thereof. In another aspect, ameganuclease provided herein edits a polynucleotide encoding apolypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255. In another aspect, a meganuclease providedherein edits a polynucleotide encoding a polypeptide having at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence similarity to a polypeptide selected from the group consistingof SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197,199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255.

The engineering of meganucleases can be more challenging than that ofZFNs and TALENs because the DNA recognition and cleavage functions ofmeganucleases are intertwined in a single domain. Specialized methods ofmutagenesis and high-throughput screening have been used to create novelmeganuclease variants that recognize unique sequences and possessimproved nuclease activity.

ZFNs are synthetic proteins consisting of an engineered zinc fingerDNA-binding domain fused to the cleavage domain of the FokI restrictionendonuclease. ZFNs can be designed to cleave almost any long stretch ofdouble-stranded DNA for modification of the zinc finger DNA-bindingdomain. ZFNs form dimers from monomers composed of a non-specific DNAcleavage domain of FokI endonuclease fused to a zinc finger arrayengineered to bind a target DNA sequence.

The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-fingerarrays. The amino acids at positions −1, +2, +3, and +6 relative to thestart of the zinc finger ∞-helix, which contribute to site-specificbinding to the target DNA, can be changed and customized to fit specifictarget sequences. The other amino acids form the consensus backbone togenerate ZFNs with different sequence specificities. Rules for selectingtarget sequences for ZFNs are known in the art.

The FokI nuclease domain requires dimerization to cleave DNA andtherefore two ZFNs with their C-terminal regions are needed to bindopposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFNmonomer can cute the target site if the two-ZF-binding sites arepalindromic. The term ZFN, as used herein, is broad and includes amonomeric ZFN that can cleave double stranded DNA without assistancefrom another ZFN. The term ZFN is also used to refer to one or bothmembers of a pair of ZFNs that are engineered to work together to cleaveDNA at the same site.

Without being limited by any scientific theory, because the DNA-bindingspecificities of zinc finger domains can in principle be re-engineeredusing one of various methods, customized ZFNs can theoretically beconstructed to target nearly any gene sequence. Publicly availablemethods for engineering zinc finger domains include Context-dependentAssembly (CoDA), Oligomerized Pool Engineering (OPEN), and ModularAssembly.

In one aspect, a ZFN provided herein edits a polynucleotide having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to a polynucleotide selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225-228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, and fragments thereof. In another aspect, a ZFN providedherein edits a polynucleotide encoding a polypeptide having at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to a polypeptide selected from the group consisting ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199,201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255. Inanother aspect, a ZFN provided herein edits a polynucleotide encoding apolypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence similarity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255.

TALENs are artificial restriction enzymes generated by fusing thetranscription activator-like effector (TALE) DNA binding domain to aFokI nuclease domain. When each member of a TALEN pair binds to the DNAsites flanking a target site, the FokI monomers dimerize and cause adouble-stranded DNA break at the target site.

The term TALEN, as used herein, is broad and includes a monomeric TALENthat can cleave double stranded DNA without assistance from anotherTALEN. The term TALEN is also used to refer to one or both members of apair of TALENs that work together to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bindpractically any DNA sequence. TALE proteins are DNA-binding domainsderived from various plant bacterial pathogens of the genus Xanthomonas.The X pathogens secrete TALEs into the host plant cell during infection.The TALE moves to the nucleus, where it recognizes and binds to aspecific DNA sequence in the promoter region of a specific DNA sequencein the promoter region of a specific gene in the host genome. TALE has acentral DNA-binding domain composed of 13-28 repeat monomers of 33-34amino acids. The amino acids of each monomer are highly conserved,except for hypervariable amino acid residues at positions 12 and 13. Thetwo variable amino acids are called repeat-variable diresidues (RVDs).The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognizeadenine, thymine, cytosine, and guanine/adenine, respectively, andmodulation of RVDs can recognize consecutive DNA bases. This simplerelationship between amino acid sequence and DNA recognition has allowedfor the engineering of specific DNA binding domains by selecting acombination of repeat segments containing the appropriate RVDs.

Besides the wild-type FokI cleavage domain, variants of the FokIcleavage domain with mutations have been designed to improve cleavagespecificity and cleavage activity. The FokI domain functions as a dimer,requiring two constructs with unique DNA binding domains for sites inthe target genome with proper orientation and spacing. Both the numberof amino acid residues between the TALEN DNA binding domain and the FokIcleavage domain and the number of bases between the two individual TALENbinding sites are parameters for achieving high levels of activity.

The relationship between amino acid sequence and DNA recognition of theTALE binding domain allows for designable proteins. Software programssuch as DNA Works can be used to design TALE constructs. Other methodsof designing TALE constructs are known to those of skill in the art. SeeDoyle et al, Nucleic Acids Research (2012) 40: W117-122.; Cermak et al.,Nucleic Acids Research (2011). 39:e82; andtale-nt.cac.cornell.edu/about.

In one aspect, a TALEN provided herein edits a polynucleotide having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to a polynucleotide selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225-228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, and fragments thereof. In another aspect, a TALEN providedherein edits a polynucleotide encoding a polypeptide having at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to a polypeptide selected from the group consisting ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199,201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255. Inanother aspect, a TALEN provided herein edits a polynucleotide encodinga polypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence similarity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255.

A CRISPR/Cas9 system or a CRISPR/Cpf1 system are alternatives to theFokI-based methods ZFN and TALEN. The CRISPR systems are based onRNA-guided engineered nucleases that use complementary base pairing torecognize DNA sequences at target sites.

CRISPR/Cas9 systems are part of the adaptive immune system of bacteriaand archaea, protecting them against invading nucleic acids such asviruses by cleaving the foreign DNA in a sequence-dependent manner. Theimmunity is acquired by the integration of short fragments of theinvading DNA known as spacers between two adjacent repeats at theproximal end of a CRISPR locus. The CRISPR arrays, including thespacers, are transcribed during subsequent encounters with invasive DNAand are processed into small interfering CRISPR RNAs (crRNAs)approximately 40 nt in length, which combine with the trans-activatingCRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease. Thiscleaves homologous double-stranded DNA sequences known as protospacersin the invading DNA. A prerequisite for cleavage is the presence of aconserved protospacer-adjacent motif (PAM) downstream of the target DNA,which usually has the sequence 5-NGG-3 but less frequently NAG.Specificity is provided by the so-called “seed sequence” approximately12 bases upstream of the PAM, which must match between the RNA andtarget DNA. Cpf1 acts in a similar manner to Cas9, but Cpf1 does notrequire a tracrRNA.

In one aspect, an engineered guide RNA provided herein guides a Cas9 orCpf1 nuclease to a polynucleotide having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to apolynucleotide selected from the group consisting of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83-160, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, and fragments thereof.In another aspect, an engineered guide RNA provided herein guides a Cas9or Cpf1 nuclease to a polynucleotide encoding a polypeptide having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193,195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,and 255. In another aspect, an engineered guide RNA provided hereinguides a Cas9 or Cpf1 nuclease to a polynucleotide encoding apolypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence similarity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255.

In one aspect, a Cas9 or a Cpf1 nuclease provided herein cleaves apolynucleotide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to a polynucleotide selectedfrom the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, and fragments thereof. In another aspect,a Cas9 or a Cpf1 nuclease provided herein cleaves a polynucleotideencoding a polypeptide having at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to a polypeptideselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, and 255. In another aspect, a Cas9 or aCpf1 nuclease provided herein cleaves a polynucleotide encoding apolypeptide having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence similarity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255.

In one aspect, a mutagenesis system provided herein (e.g., chemicalmutagenesis, irradiation mutagenesis, transposon mutagenesis,Agrobacterium-mediated transformation, a meganuclease, a ZFN, a TALEN, aCRISPR/Cas9 system, a CRISPR/Cpf1 system), or a combination ofmutagenesis systems provided herein, is used in a method to introduceone or more mutations to a tobacco gene that is natively expressed in atleast one tobacco axillary meristem cell.

In still another aspect, a modified tobacco plant provided hereinfurther comprises one or more mutations in one or more loci encoding anicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) that conferreduced amounts of nornicotine (See U.S. Pat. Nos. 8,319,011; 8,124,851;9,187,759; 9,228,194; 9,228,195; 9,247,706) compared to control plantlacking one or more mutations in one or more loci encoding a nicotinedemethylase. In another aspect, a tobacco plant provided herein furthercomprises one or more mutations in a Nic1 locus, a Nic2 locus, or both,which confer reduced amounts of nicotine compared to a control plantlacking one or more mutations in a Nic1 locus, a Nic2 locus, or both.

In one aspect, recombinant DNA constructs or expression cassettesprovided herein comprise a promoter selected from the group consistingof a constitutive promoter, an inducible promoter, and atissue-preferred promoter (for example, without being limiting, aleaf-specific promoter, a root-specific promoter, or a meristem-specificpromoter).

In one aspect, a promoter provided herein is an axillary bud-specificpromoter. In one aspect, a promoter provided herein is an axillarymeristem-specific promoter. In one aspect, an axillary meristem-specificpromoter provided herein is functional or preferentially functional inan L1 layer, an L2 layer, an L3 layer, or a combination thereof. Dicotshoot apical and axillary meristems comprise three distinct cell layers:the L1 layer (outermost layer), the L2 layer (middle layer), and the L3layer (innermost layer). The L1 and L2 layers make up the tunica, andthey divide anticlinally (the division plane is perpendicular to thesurface of the meristem). The L3 layer, or corpus, divides in alldirections. The L1 layer eventually gives rise to epidermal tissue; theL2 layer gives rise to ground tissue (e.g., parenchyma, collenchyma,sclerenchyma);and the L3 layer typically gives rise to vascular tissue(e.g., xylem, phloem)

Shoot apical and axillary meristems can also be divided into threezones: a central zone, a peripheral zone, and a rib zone. Cells from thecentral zone, comprising parts of the L1, L2, and L3 layers at the peakof the meristem, serve to organize and maintain the meristem; thecentral zone comprises pluripotent stem cells. The peripheral zonesurrounds the central zone and will form organs (e.g., leaf primordia,flower primordia) and undergo morphogenesis; this zone comprises highmitotic activity. The rib zone, or rib meristem, is positioned below thecentral zone; the rib zone gives rise to stem and vasculature tissue. Inone aspect, an axillary meristem-specific promoter provided herein isfunctional or preferentially functional in a central zone, a peripheralzone, a rib zone, or a combination thereof.

In one aspect, an axillary bud-specific promoter comprises apolynucleotide sequence having at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to apolynucleotide selected from the group consisting of SEQ ID NOs:113-118, 148-160, 204 and fragments thereof. In another aspect, anaxillary meristem-specific promoter comprises a polynucleotide sequencehaving at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to a polynucleotide selectedfrom the group consisting of SEQ ID NOs: 113-118, 148-160, 204 andfragments thereof. In still another aspect, a promoter active in an L1layer, an L2 layer, an L3 layer, or a combination thereof comprises apolynucleotide sequence selected from the group consisting of SEQ IDNOs: 113-118, 148-160, 204 and a fragment thereof. In another aspect, apromoter active in a central zone, a peripheral zone, a rib zone, or acombination thereof comprises a polynucleotide sequence selected fromthe group consisting of SEQ ID NOs: 113-118, 148-160, 204 and a fragmentthereof. In one aspect, a promoter fragment provided herein has a lengthof at least 50, at least 75, at least 100, at least 200, at least 300,at least 400, at least 500, at least 600, at least 700, at least 800, atleast 900, at least 1000, at least 1250, at least 1500, at least 1750,at least 2000, at least 2500, at least 3000, at least 3500, at least4000, at least 4500, or at least 4999 nucleotides. In another aspect, apromoter fragment provided herein has a length of between 50 and 200,between 100 and 200, between 100 and 300, between 100 and 400, between100 and 500, between 100 and 600, between 100 and 700, between 100 and800, 100 and 900, between 100 and 1000, between 100 and 2000, between200 and 300, between 200 and 400, between 200 and 500, between 200 and600, between 200 and 700, between 200 and 800, between 200 and 900,between 200 and 1000, between 200 and 2000, between 200 and 2500,between 200 and 3000, between 500 and 1000, between 500 and 1500,between 500 and 2000, between 500 and 2500, between 500 and 3000,between 1000 and 2000, between 1000 and 3000, between 1500 and 2000,between 1500 and 2500, between 1500 and 3000, between 2000 and 3000nucleotides, between 100 and 3500, between 100 and 4000, between 100 and4500, between 100 and 4999, between 500 and 3500, between 500 and 4000,between 500 and 4500, between 500 and 4999, between 1000 and 3500,between 1000 and 4000, between 1000 and 4500, between 1000 and 4999,between 2000 and 3500, between 2000 and 4000, between 2000 and 4500,between 2000 and 4999, between 3000 and 3500, between 3000 and 4000,between 3000 and 4500, between 3000 and 4999, between 3500 and 4000,between 3500 and 4500, between 3500 and 4999, between 4000 and 4500,between 4000 and 4999, or between 4500 and 4999 nucleotides.

In one aspect, a recombinant DNA construct of the present disclosurecomprises a polynucleotide selected from the group consisting of SEQ IDNOs: 113-118, 148-160, 204 and a fragment thereof operably linked to apolynucleotide encoding a polypeptide having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to apolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, a recombinant DNA construct of the present disclosurecomprises a polynucleotide selected from the group consisting of SEQ IDNOs: 113-118, 148-160, 204 and a fragment thereof operably linked to apolynucleotide encoding a polypeptide having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence similarity to apolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, a recombinant DNA construct of the present disclosurecomprises a polynucleotide selected from the group consisting of SEQ IDNOs: 113-118, 148-160, 204 and a fragment thereof operably linked to apolynucleotide having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% identity to apolynucleotide selected from the group consisting of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83-160, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, and 225-228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, and fragments thereof.

In one aspect, a tobacco plant, or part thereof, of the presentdisclosure comprises a heterologous promoter having at least 90%sequence identity to a polynucleotide selected from the group consistingof SEQ ID NOs: 113-118, 148-160, 204, and fragments thereof operablylinked to a polynucleotide encoding an auxin biosynthesis protein or anauxin transport protein.

Exemplary constitutive promoters include the core promoter of the Rsyn7promoter and other constitutive promoters disclosed in U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like.

Exemplary chemical-inducible promoters include the tobacco PR-1apromoter, which is activated by salicylic acid. Other chemical-induciblepromoters of interest include steroid-responsive promoters (see, forexample, the glucocorticoid-inducible promoter in Schena et al. (1991)Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998)Plant J. 14(2):247-257) and tetracycline-inducible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156). Additional exemplary promoters that canbe used herein are those responsible for heat-regulated gene expression,light-regulated gene expression (for example, the pea rbcS-3A; the maizerbcS promoter; the chlorophyll alb-binding protein gene found in pea; orthe Arabssu promoter), hormone-regulated gene expression (for example,the abscisic acid (ABA) responsive sequences from the Em gene of wheat;the ABA-inducible HVA1 and HVA22, and rd29A promoters of barley andArabidopsis; and wound-induced gene expression (for example, of wunl),organ specific gene expression (for example, of the tuber-specificstorage protein gene; the 23-kDa zein gene from maize described by; orthe French bean (β-phaseolin gene), or pathogen-inducible promoters (forexample, the PR-1, prp-1, or (β-1,3 glucanase promoters, thefungal-inducible wirla promoter of wheat, and the nematode-induciblepromoters, TobRB7-5A and Hing-1, of tobacco arid parsley, respectively).

Additional exemplary tissue-preferred promoters include those disclosedin Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505.

As used herein, “operably linked” refers to a functional linkage betweentwo or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (e.g., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.In one aspect, a promoter provided herein is operably linked to apolynucleotide selected from the group consisting of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83-160, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, and fragments thereof.In another aspect, a promoter provided herein is operably linked to apolynucleotide encoding a polypeptide selected from the group consistingof SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197,199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255.

As used herein, “heterologous” refers to a sequence that originates froma foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. The term also is applicable to nucleicacid constructs, also referred to herein as “polynucleotide constructs”or “nucleotide constructs.” In this manner, a “heterologous” nucleicacid construct is intended to mean a construct that originates from aforeign species, or, if from the same species, is substantially modifiedfrom its native form in composition and/or genomic locus by deliberatehuman intervention. Heterologous nucleic acid constructs include, butare not limited to, recombinant nucleotide constructs that have beenintroduced into a plant or plant part thereof, for example, viatransformation methods or subsequent breeding of a transgenic plant withanother plant of interest.

In one aspect, a modified plant, seed, plant component, plant cell, orplant genome provided herein comprises a heterologous promoter operablylinked to a polynucleotide encoding a polypeptide having at least 40%,at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a polypeptide selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191,193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, and 255. In another aspect, a recombinant DNA construct providedherein comprises a heterologous promoter operably linked to apolynucleotide encoding a polypeptide having at least 40%, at least 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193,195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,and 255.

Enhancer elements are regions of DNA that can be bound by proteins toactivate RNA transcription. In one aspect, a promoter sequence providedherein is operably linked to an enhancer element. In another aspect, apolynucleotide selected from the group consisting of SEQ ID NOs:113-118, 148-160, 204, and a fragment thereof is operably linked to anenhancer element. In one aspect, an enhancer element provided herein isat least 10, at least 25, at least 50, at least 75, at least 100, atleast 150, at least 200, at least 250, at least 300, at least 350, atleast 400, at least 450, at least 500, at least 550, at least 600, atleast 650, at least 700, at least 750, at least 800, at least 850, atleast 900, at least 950, at least 1000, at least 1100, at least 1200, atleast 1300, at least 1400, at least 1500, at least 1750, at least 2000,at least 2500, at least 3000, at least 3500, at least 4000, at least4500, or at least 5000 nucleotides in length. In one aspect, an enhancerelement provided herein is a CsVMV promoter.

Many gene promoters contain cis-regulatory elements that function toregulate gene transcription. Cis-regulatory elements often function byserving as binding sites for transcription factors. In one aspect, apromoter provided herein comprises at least 1, at least 2, at least 3,at least 4, at least 5, or at least 6 cis regulatory elements selectedfrom the group consisting of a bud dormancy element (BDE), an axillarybud growth UP1 element, an axillary bud growth UP2 element, a sucroseresponsive element (SRE), a sugar repressive element (SURE), and a budactivation or TCP-binding element (BAE). In another aspect, arecombinant nucleotide provided herein comprises a promoter, where thepromoter comprises at least 1, at least 2, at least 3, at least 4, atleast 5, or at least 6 cis elements selected from the group consistingof a bud dormancy element (BDE), an axillary bud growth UP1 element, anaxillary bud growth UP2 element, a sucrose responsive element (SURE), asugar repressive element (SRE), and a bud activation or TCP-bindingelement (BAE).

In one aspect, a promoter provided herein comprises at least 1, at least2, at least 3, at least 4, at least 5, or at least 6 cis-regulatoryelements within 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600,2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800,3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000,7500, or 10,000 nucleotides of a transcriptional start site. In anotheraspect, a promoter provided herein comprises at least 1, at least 2, atleast 3, at least 4, at least 5, or at least 6 cis-regulatory elementswithin 1-10,000, 1-7500, 1-5000, 1-4900, 1-4800, 1-4700, 1-4600, 1-4500,1-4400, 1-4300, 1-4200, 1-4100, 1-4000, 1-3900, 1-3800, 1-3700, 1-3600,1-3500, 1-3400, 1-3300, 1-3200, 1-3100, 1-3000, 1-2900, 1-2800, 1-2700,1-2600, 1-2500, 1-2400, 1-2300, 1-2200, 1-2100, 1-2000, 1-1900, 1-1800,1-1700, 1-1600, 1-1500, 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, 1-900,1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-75, 1-50,1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 5-10,000, 5-7500, 5-5000,5-4900, 5-4800, 5-4700, 5-4600, 5-4500, 5-4400, 5-4300, 5-4200, 5-4100,5-4000, 5-3900, 5-3800, 5-3700, 5-3600, 5-3500, 5-3400, 5-3300, 5-3200,5-3100, 5-3000, 5-2900, 5-2800, 5-2700, 5-2600, 5-2500, 5-2400, 5-2300,5-2200, 5-2100, 5-2000, 5-1900, 5-1800, 5-1700, 5-1600, 5-1500, 5-1400,5-1300, 5-1200, 5-1100, 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500,5-400, 5-300, 5-200, 5-100, 5-75, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25,5-20, 20-10,000, 20-7500, 20-5000, 20-4900, 20-4800, 20-4700, 20-4600,20-4500, 20-4400, 20-4300, 20-4200, 20-4100, 20-4000, 20-3900, 20-3800,20-3700, 20-3600, 20-3500, 20-3400, 20-3300, 20-3200, 20-3100, 20-3000,20-2900, 20-2800, 20-2700, 20-2600, 20-2500, 20-2400, 20-2300, 20-2200,20-2100, 20-2000, 20-1900, 20-1800, 20-1700, 20-1600, 20-1500, 20-1400,20-1300, 20-1200, 20-1100, 20-1000, 20-900, 20-800, 20-700, 20-600,20-500, 20-400, 20-300, 20-200, 20-100, 20-75, 20-50, 20-45, 20-40,20-35, 20-30, 20-25, 50-10,000, 50-7500, 50-5000, 50-4900, 50-4800,50-4700, 50-4600, 50-4500, 50-4400, 50-4300, 50-4200, 50-4100, 50-4000,50-3900, 50-3800, 50-3700, 50-3600, 50-3500, 50-3400, 50-3300, 50-3200,50-3100, 50-3000, 50-2900, 50-2800, 50-2700, 50-2600, 50-2500, 50-2400,50-2300, 50-2200, 50-2100, 50-2000, 50-1900, 50-1800, 50-1700, 50-1600,50-1500, 50-1400, 50-1300, 50-1200, 50-1100, 50-1000, 50-900, 50-800,50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75,100-10,000, 100-7500, 100-5000, 100-4900, 100-4800, 100-4700, 100-4600,100-4500, 100-4400, 100-4300, 100-4200, 100-4100, 100-4000, 100-3900,100-3800, 100-3700, 100-3600, 100-3500, 100-3400, 100-3300, 100-3200,100-3100, 100-3000, 100-2900, 100-2800, 100-2700, 100-2600, 100-2500,100-2400, 100-2300, 100-2200, 100-2100, 100-2000, 100-1900, 100-1800,100-1700, 100-1600, 100-1500, 100-1400, 100-1300, 100-1200, 100-1100,100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300,100-200, 500-10,000, 500-7500, 500-5000, 500-4900, 500-4800, 500-4700,500-4600, 500-4500, 500-4400, 500-4300, 500-4200, 500-4100, 500-4000,500-3900, 500-3800, 500-3700, 500-3600, 500-3500, 500-3400, 500-3300,500-3200, 500-3100, 500-3000, 500-2900, 500-2800, 500-2700, 500-2600,500-2500, 500-2400, 500-2300, 500-2200, 500-2100, 500-2000, 500-1900,500-1800, 500-1700, 500-1600, 500-1500, 500-1400, 500-1300, 500-1200,500-1100, 500-1000, 500-900, 500-800, 500-700, or 500-600 nucleotides ofa transcriptional start site.

In one aspect, a promoter provided herein is functional in an axillarybud cell and comprises at least 1, at least 2, at least 3, at least 4,at least 5, or at least 6 cis-regulatory elements selected from thegroup consisting of a bud dormancy element (BDE), an axillary bud growthUP1 element, an axillary bud growth UP2 element, a sucrose responsiveelement (SURE), a sugar repressive element (SRE), and a bud activationor TCP-binding element (BAE).

Also provided herein are the transformation of tobacco plants withrecombinant constructs or expression cassettes described herein usingany suitable transformation methods known in the art. Methods forintroducing polynucleotide sequences into tobacco plants are known inthe art and include, but are not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.“Stable transformation” refers to transformation where the nucleotideconstruct of interest introduced into a plant integrates into a genomeof the plant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a sequence isintroduced into the plant and is only temporally expressed or is onlytransiently present in the plant.

In one aspect, methods and compositions provided herein comprise theintroduction of one or more polynucleotides into one or more plantcells. In one aspect, a plant genome provided herein is modified toinclude an introduced polynucleotide or recombinant DNA construct. Asused herein, “plant genome” refers to a nuclear genome, a mitochondrialgenome, or a plastid (e.g., chloroplast) genome of a plant cell. Inanother aspect, a polynucleotide provided herein is integrated into anartificial chromosome. In one aspect, an artificial chromosomecomprising a polynucleotide provided herein is integrated into a plantcell.

In one aspect, a modified plant, seed, plant component, plant cell, orplant genome provided herein comprises one or more transgenes. In oneaspect, a transgene provided herein suppresses suckering in a plant. Inanother aspect, a transgene provided herein suppresses topping-inducedsuckering in a plant. In still another aspect, a transgene providedherein suppresses suckering in a plant prior to topping. As used herein,a “transgene” refers to a polynucleotide that has been transferred intoa genome by any method known in the art. In one aspect, a transgene isan exogenous polynucleotide. In one aspect, a transgene is an endogenouspolynucleotide that is integrated into a new genomic locus where it isnot normally found.

In one aspect, transgenes provided herein comprise a recombinant DNAconstruct. In one aspect, recombinant DNA constructs or expressioncassettes provided herein can comprise a selectable marker gene for theselection of transgenic cells. Selectable marker genes include, but arenot limited to, genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, triazolopyrimidines, sulfonylurea (e.g., chlorsulfuronand sulfometuron methyl), and 2,4-dichlorophenoxyacetate (2,4-D).Additional selectable markers include phenotypic markers such asβ-galactosidase and fluorescent proteins such as green fluorescentprotein (GFP).

In one aspect, methods and compositions provided herein comprise avector. As used herein, the terms “vector” or “plasmid” are usedinterchangeably and refer to a circular, double-stranded DNA moleculethat is physically separate from chromosomal DNA. In one aspect, aplasmid or vector used herein is capable of replication in vivo. A“transformation vector,” as used herein, is a plasmid that is capable oftransforming a plant cell. In an aspect, a plasmid provided herein is abacterial plasmid. In another aspect, a plasmid provided herein is anAgrobacterium Ti plasmid or derived from an Agrobacterium Ti plasmid.

In one aspect, a plasmid or vector provided herein is a recombinantvector. As used herein, the term “recombinant vector” refers to a vectorformed by laboratory methods of genetic recombination, such as molecularcloning. In another aspect, a plasmid provided herein is a syntheticplasmid. As used herein, a “synthetic plasmid” is an artificiallycreated plasmid that is capable of the same functions (e.g.,replication) as a natural plasmid (e.g., Ti plasmid). Without beinglimited, one skilled in the art can create a synthetic plasmid de novovia synthesizing a plasmid by individual nucleotides, or by splicingtogether nucleic acid molecules from different pre-existing plasmids.

Vectors are commercially available or can be produced by recombinant DNAtechniques routine in the art. In one aspect, a vector provided hereincomprises all or part of SEQ ID NO: 112. A vector containing a nucleicacid can have expression elements operably linked to such a nucleicacid, and further can include sequences such as those encoding aselectable marker (e.g., an antibiotic resistance gene). A vectorcontaining a nucleic acid can encode a chimeric or fusion polypeptide(i.e., a polypeptide operatively linked to a heterologous polypeptide,which can be at either the N-terminus or C-terminus of the polypeptide).Representative heterologous polypeptides are those that can be used inpurification of the encoded polypeptide (e.g., 6× His tag (SEQ ID NO:256), glutathione S-transferase (GST)).

Suitable methods of introducing polynucleotides (e.g., transgenes,recombinant vectors, recombinant DNA constructs, expression constructs)into plant cells of the present disclosure include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation(Shillito et al. (1987) Meth. Enzymol. 153:313-336; Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,104,310, 5,149,645, 5,177,010,5,231,019, 5,463,174, 5,464,763, 5,469,976, 4,762,785, 5,004,863,5,159,135, 5,563,055, and 5,981,840), direct gene transfer (Paszkowskiet al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration(see, for example, U.S. Pat. Nos. 4,945,050, 5,141,131, 5,886,244,5,879,918, and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, andOrgan Culture Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674 (soybean);McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer andMcMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singhet al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation). In one aspect, a bacterialcell provided herein comprises a recombinant DNA construct orrecombinant vector provided herein.

In another aspect, recombinant constructs or expression cassettesprovided herein may be introduced into plants by contacting plants witha virus or viral nucleic acids. Generally, such methods involveincorporating an expression cassette of the present disclosure within aviral DNA or RNA molecule. It is recognized that promoters for use inthe expression cassettes provided herein also encompass promotersutilized for transcription by viral RNA polymerases. Methods forintroducing polynucleotides into plants and expressing a protein encodedtherein, involving viral DNA or RNA molecules, are known in the art.See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology5:209-221.

Any plant tissue that can be subsequently propagated using clonalmethods, whether by organogenesis or embryogenesis, may be transformedwith a recombinant construct or an expression cassette provided herein.By “organogenesis” in intended the process by which shoots and roots aredeveloped sequentially from meristematic centers. By “embryogenesis” isintended the process by which shoots and roots develop together in aconcerted fashion (not sequentially), whether from somatic cells orgametes. Exemplary tissues that are suitable for various transformationprotocols described herein include, but are not limited to, callustissue, existing meristematic tissue (e.g., apical meristems, axillarybuds, and root meristems) and induced meristem tissue (e.g., cotyledonmeristem and hypocotyl meristem), hypocotyls, cotyledons, leaf disks,pollen, embryos, and the like.

In one aspect, this disclosure provides a plant or seed comprising arecombinant polynucleotide, where the recombinant polynucleotidecomprises a promoter that is functional in an L1 layer, an L2 layer, anL3 region, a rib zone, a central zone, a peripheral zone, or anycombination thereof, which is operably linked to a structural nucleicacid molecule comprising a nucleic acid sequence, where the nucleic acidsequence encodes a polypeptide having at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193,195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,and 255.

In one aspect, an auxin biosynthesis protein or an auxin transportprotein is selected from the group consisting of SEQ ID NOs: 235, 237,239, 241, 243, 245, 247, 249, 251, 253, and 255. In another aspect, anauxin biosynthesis protein or an auxin transport protein comprises atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a polypeptideselected from the group consisting of SEQ ID NOs: 235, 237, 239, 241,243, 245, 247, 249, 251, 253, and 255. In another aspect, an auxinbiosynthesis protein or an auxin transport protein comprises at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence similarity to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 235, 237, 239, 241, 243, 245,247, 249, 251, 253, and 255. In one aspect, an auxin biosynthesisprotein or an auxin transport protein is encoded by a nucleic acidsequence, where the nucleic acid sequence encodes a polypeptide havingat least 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to a polypeptideelected from the group consisting of SEQ ID NOs: 235, 237, 239, 241,243, 245, 247, 249, 251, 253, and 255.

In one aspect, an auxin biosynthesis protein or an auxin transportprotein is encoded by a polynucleotide selected from the groupconsisting of SEQ ID NOs: 234, 236, 238, 240, 242, 244, 246, 248, 250,252, and 254. In one aspect, an auxin biosynthesis protein or an auxintransport protein is encoded by a polynucleotide having at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identity to a polynucleotide selected from the groupconsisting of SEQ ID NOs: 234, 236, 238, 240, 242, 244, 246, 248, 250,252, and 254.

In one aspect, this disclosure provides a plant or seed comprising arecombinant polynucleotide, where the recombinant polynucleotidecomprises a promoter that is functional in an L1 layer, an L2 layer, anL3 region, a rib zone, a central zone, a peripheral zone, or acombination thereof, which is operably linked to a structural nucleicacid molecule comprising a nucleic acid sequence, where the nucleic acidsequence encodes an auxin biosynthesis protein or an auxin transportprotein.

In another aspect, this disclosure provides a recombinant DNA constructcomprising a promoter that is functional in an L1 layer, an L2 layer, anL3 region, a rib zone, a central zone, a peripheral zone, or acombination thereof; and a heterologous and operably linked nucleic acidsequence, where the nucleic acid sequence encodes a non-coding RNA or apolypeptide. In another aspect, this disclosure provides a recombinantDNA construct comprising a promoter that is functional in an L1 later,an L2 layer, an L3 region, a rib zone, a central zone, a peripheralzone, or a combination thereof; and a heterologous and operably linkednucleic acid sequence, where the nucleic acid sequence encodes an auxinbiosynthesis protein or an auxin transport protein.

In one aspect, this disclosure provides a recombinant DNA constructcomprising a heterologous axillary meristem-specific promoter operablylinked to a polynucleotide that encodes an auxin biosynthesis protein oran auxin transport protein.

In one aspect, this disclosure provides a method of reducing oreliminating topping-induced suckering in a tobacco plant comprisingtransforming a tobacco plant with a recombinant DNA construct comprisinga promoter expressing in an L1 layer, an L2 layer, an L3 region, a ribzone, a central zone, a peripheral zone, or a combination thereof. Inanother aspect, this disclosure provides a method comprisingtransforming a tobacco plant with a recombinant DNA construct comprisinga heterologous promoter that is functional in an L1 layer, an L2 layer,an L3 region, a rib zone, a central zone, a peripheral zone, or acombination thereof, and is operably linked to a polynucleotide that istranscribed into an RNA molecule that suppresses the level of anendogenous gene, and where the endogenous gene promotes or is requiredfor axillary meristem growth, axillary meristem maintenance, or both.

In one aspect, this disclosure provides a method for controllingtopping-induced suckering in a plant comprising transforming the plantwith a recombinant DNA construct, where the recombinant DNA constructcomprises a promoter that is operably linked to a polynucleotideencoding a polypeptide having at least 40%, at least 50%, at least 60%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a polypeptide selected from the group consisting of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201,203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, this disclosure provides a method for controllingtopping-induced suckers in a plant comprising transforming said plantwith a recombinant DNA construct, wherein said recombinant DNA constructcomprises a promoter that is operably linked to a polynucleotideencoding an auxin biosynthesis protein or an auxin transport protein.

It is understood that any modified tobacco plant of the presentdisclosure can further comprise additional agronomically desirabletraits, for example, by transformation with a genetic construct ortransgene using a technique known in the art. Without limitation, anexample of a desired trait is herbicide resistance, pest resistance,disease resistance, high yield, high grade index value, curability,curing quality, mechanical harvestability, holding ability, leafquality, height, plant maturation (e.g., early maturing, early to mediummaturing, medium maturing, medium to late maturing, or late maturing),stalk size (e.g., a small, medium, or a large stalk), or leaf number perplant (e.g., a small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves),or large (e.g., 16-21) number of leaves), or any combination. In oneaspect, reduced suckering tobacco plants or seeds provided hereincomprise one or more transgenes expressing one or more insecticidalproteins, such as, for example, a crystal protein of Bacillusthuringiensis or a vegetative insecticidal protein from Bacillus cereus,such as VIP3 (see, for example, Estruch et al. (1997) Nat.Biotechnol.15:137). In another aspect, tobacco plants provided hereinfurther comprise an introgressed trait conferring resistance to brownstem rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S.Pat. No. 5,491,081).

The level and/or activity of polypeptides provided herein may bemodulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or anRNA. For example, the polynucleotides of the invention may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984;each of which is incorporated herein by reference as if set forth in itsentirety. See also, International Patent Application Publication Nos. WO98/149350, WO 99/107865 and WO 99/125921; and Beetham et al. (1999)Proc. Natl. Acad. Sci. USA 96:8774-8778; each of which is incorporatedherein by reference as if set forth in its entirety.

The present disclosure provides compositions and methods for inhibitingthe expression or function of one or more polypeptides selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191,193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, and 255 in a plant, particularly plants of the Nicotiana tabacumgenus, including tobacco plants of various commercial varieties.

In one aspect, inhibition of the expression of one or more polypeptidesprovided herein may be obtained by RNA interference (RNAi) by expressionof a polynucleotide provided herein. In one aspect, RNAi comprisesexpressing a non-coding RNA. As used herein, a “non-coding RNA” isselected from the group consisting of a microRNA (miRNA), a smallinterfering RNA (siRNA), a trans-acting siRNA (ta-siRNA), a transfer RNA(tRNA), a ribosomal RNA (rRNA), an intron, a hairpin RNA (hpRNA), and anintron-containing hairpin RNA (ihpRNA). In one aspect, a singlenon-coding RNA provided herein inhibits the expression of at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, or more than 10 polypeptides. In oneaspect, a non-coding RNA provided herein is stably transformed into aplant genome. In another aspect, a non-coding RNA provided herein istransiently transformed into a plant genome.

In one aspect, this disclosure provides RNA molecules useful forinhibiting the expression or function or one or more polypeptidesselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, and 255. In one aspect, an RNA moleculeprovided herein is a non-coding RNA.

In another aspect, the recombinant DNA construct encodes a doublestranded RNA. Also provided are modified tobacco plants or part thereof,cured tobacco material, or tobacco products comprising these recombinantDNA constructs. In one aspect, these transgenic plants, cured tobaccomaterial, or tobacco products comprise reduced suckering compared to acontrol tobacco plant without the recombinant DNA construct. Furtherprovided are methods of reducing sucker growth of a tobacco plant, themethod comprising transforming a tobacco plant with any of theserecombinant DNA constructs.

In one aspect, a tobacco plant or part thereof provided herein comprisesa heterologous promoter operably linked to a polynucleotide that encodesa non-coding RNA molecule, where the non-coding RNA molecule is capableof binding to an RNA encoding a polypeptide having at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a polypeptide selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 161-185, 187, 189, 191, 193,195, 197, 199, 224, 229, 231, 233, 245, 247, 249, 251, 253, and 255,andwhere the non-coding RNA molecule suppresses the expression of thepolypeptide.

As used herein, the terms “suppress,” “inhibit,” “inhibition,” and“inhibiting” are defined as any method known in the art or describedherein that decreases the expression or function of a gene product ofinterest (e.g., an mRNA, a protein, a non-coding RNA). “Inhibition” canbe in the context of a comparison between two plants, for example, amodified plant versus a control plant. Alternatively, inhibition ofexpression or function of a target gene product can be in the context ofa comparison between plant cells, organelles, organs, tissues, or plantcomponents within the same plant or between different plants, andincludes comparisons between developmental or temporal stages within thesame plant or plant component or between plants or plant components.“Inhibition” includes any relative decrement of function or productionof a gene product of interest, up to and including complete eliminationof function or production of that gene product. The term “inhibition”encompasses any method or composition that down-regulates translationand/or transcription of the target gene product or functional activityof the target gene product.

The term “inhibitory sequence” encompasses any polynucleotide orpolypeptide sequence capable of inhibiting the expression or function ofa gene in a plant, such as full-length polynucleotide or polypeptidesequences, truncated polynucleotide or polypeptide sequences, fragmentsof polynucleotide or polypeptide sequences, variants of polynucleotideor polypeptide sequences, sense-oriented nucleotide sequences,antisense-oriented nucleotide sequences, the complement of a sense- orantisense-oriented nucleotide sequence, inverted regions of nucleotidesequences, hairpins of nucleotide sequences, double-stranded nucleotidesequences, single-stranded nucleotide sequences, combinations thereof,and the like. The term “polynucleotide sequence” includes sequences ofRNA, DNA, chemically modified nucleic acids, nucleic acid analogs,combinations thereof, and the like.

Inhibitory sequences are designated herein by the name of the targetgene product. Thus, as a non-limiting example, an “NTH15 inhibitorysequence” refers to an inhibitory sequence that is capable of inhibitingthe expression of an NTH15 locus in a plant, for example, at the levelof transcription and/or translation, or which is capable of inhibitingthe function of a gene product. When the phrase “capable of inhibiting”is used in the context of a polynucleotide inhibitory sequence, it isintended to mean that the inhibitory sequence itself exerts theinhibitory effect; or, where the inhibitory sequence encodes aninhibitory nucleotide molecule (for example, hairpin RNA, miRNA, ordouble-stranded RNA polynucleotides), or encodes an inhibitorypolypeptide (e.g., a polypeptide that inhibits expression or function ofthe target gene product), following its transcription (for example, inthe case of an inhibitory sequence encoding a hairpin RNA, miRNA, ordouble-stranded RNA polynucleotide) or its transcription and translation(in the case of an inhibitory sequence encoding an inhibitorypolypeptide), the transcribed or translated product, respectively,exerts the inhibitory effect on the target gene product (e.g., inhibitsexpression or function of the target gene product).

An inhibitory sequence provided herein can be a sequence triggering genesilencing via any silencing pathway or mechanism known in the art,including, but not limited to, sense suppression/co-suppression,antisense suppression, double-stranded RNA (dsRNA) interference, hairpinRNA interference and intron-containing hairpin RNA interference,amplicon-mediated interference, ribozymes, small interfering RNA,artificial or synthetic microRNA, and artificial trans-acting siRNA. Aninhibitory sequence may range from at least about 20 nucleotides, atleast about 50 nucleotides, at least about 70 nucleotides, at leastabout 100 nucleotides, at least about 150 nucleotides, at least about200 nucleotides, at least about 250 nucleotides, at least about 300nucleotides, at least about 350 nucleotides, at least about 400nucleotides, and up to the full-length polynucleotide encoding theproteins of the present disclosure, depending upon the desired outcome.In one aspect, an inhibitory sequence can be a fragment of between about50 and about 400 nucleotides, between about 70 and about 350nucleotides, between about 90 and about 325 nucleotides, between about90 and about 300 nucleotides, between about 90 and about 275nucleotides, between about 100 and about 400 nucleotides, between about100 and about 350 nucleotides, between about 100 and about 325nucleotides, between about 100 and about 300 nucleotides, between about125 and about 300 nucleotides, or between about 125 and about 275nucleotides in length.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a promoter that is functional in a tobacco cell andoperably linked to a polynucleotide that encodes an RNA molecule capableof binding to an RNA encoding a polypeptide having an amino acidsequence at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 187,189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, and fragments thereof. In one aspect, thepresent disclosure provides a recombinant DNA construct comprising apromoter that is functional in a tobacco cell and operably linked to apolynucleotide having at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to a polynucleotideselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, and fragments thereof.

In one aspect, the present disclosure provides a recombinant DNAconstruct comprising a promoter that is functional in a tobacco cell andoperably linked to a polynucleotide that encodes an RNA molecule capableof binding to an RNA encoding a polypeptide having an amino acidsequence at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 187,189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, and fragments thereof, and where the RNAmolecule suppresses the expression of the polypeptide. In one aspect,the present disclosure provides a recombinant DNA construct comprising apromoter that is functional in a tobacco cell and operably linked to apolynucleotide having at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to a polynucleotideselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, and fragments thereof, and where theRNA molecule suppresses the expression of the polynucleotide.

In one aspect, this disclosure provides a recombinant DNA constructcomprising a heterologous axillary meristem-specific promoter operablylinked to a polynucleotide that encodes a non-coding RNA molecule, wherethe non-coding RNA molecule is capable of binding to an RNA encoding apolypeptide having at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78,161-185, 187, 189, 191, 197, 199, 224, 229, 231, 233, 245, 247, 249,251, 253, and 255, and where the non-coding RNA molecule suppresses theexpression of the polypeptide.

In one aspect, this disclosure provides a method for controllingtopping-induced suckering in a plant comprising transforming the plantwith a recombinant DNA construct, where the recombinant DNA constructcomprises a heterologous promoter that is functional in an L1 layer, anL2 layer, an L3 region, a rib zone, a central zone, a peripheral zone,or a combination thereof, and where the promoter is operably linked to apolynucleotide that encodes a non-coding RNA molecule, where thenon-coding RNA molecule is capable of binding to an RNA encoding apolypeptide having at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78,161-185, 187, 189, 191, 197, 199, 224, 229, 231, 233, 245, 247, 249,251, 253, and 255, and where the non-coding RNA molecule suppresses theexpression of the polypeptide.

MicroRNAs (miRNAs) are non-protein coding RNAs, generally of betweenabout 19 to about 25 nucleotides (commonly about 20-24 nucleotides inplants), that guide cleavage in trans of target transcripts, negativelyregulating the expression of genes involved in various regulation anddevelopment pathways (Bartel (2004) Cell, 116:281-297). In some cases,miRNAs serve to guide in-phase processing of siRNA primary transcripts(see Allen et al. (2005) Cell, 121:207-221).

Many microRNA genes (MIR genes) have been identified and made publiclyavailable in a database (“miRBase”, available on line atmicrorna.sanger.ac.uk/sequences; also see Griffiths-Jones et al. (2003)Nucleic Acids Res., 31:439-441). MIR genes have been reported to occurin intergenic regions, both isolated and in clusters in the genome, butcan also be located entirely or partially within introns of other genes(both protein-coding and non-protein-coding). For a recent review ofmiRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol.,6:376-385. Transcription of MIR genes can be, at least in some cases,under promotional control of a MIR gene's own promoter. The primarytranscript, termed a “pri-miRNA”, can be quite large (several kilobases)and can be polycistronic, containing one or more pre-miRNAs (fold-backstructures containing a stem-loop arrangement that is processed to themature miRNA) as well as the usual 5′ “cap” and polyadenylated tail ofan mRNA. See, for example, FIG. 1 in Kim (2005) Nature Rev. Mol. Cell.Biol., 6:376-385.

Maturation of a mature miRNA from its corresponding precursors(pri-miRNAs and pre-miRNAs) differs significantly between animals andplants. For example, in plant cells, microRNA precursor molecules arebelieved to be largely processed to the mature miRNA entirely in thenucleus, whereas in animal cells, the pri-miRNA transcript is processedin the nucleus by the animal-specific enzyme Drosha, followed by exportof the pre-miRNA to the cytoplasm where it is further processed to themature miRNA. Mature miRNAs in plants are typically 21 nucleotides inlength. For a recent review of miRNA biogenesis in both plants andanimals, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385.Additional reviews on miRNA biogenesis and function are found, forexample, in Bartel (2004) Cell, 116:281-297; Murchison and Hannon (2004)Curr. Opin. Cell Biol., 16:223-229; and Dugas and Bartel (2004) Curr.Opin. Plant Biol., 7:512-520.

Transgenic expression of miRNAs (whether a naturally occurring sequenceor an artificial sequence) can be employed to regulate expression of themiRNA's target gene or genes. Inclusion of a miRNA recognition site in atransgenically expressed transcript is also useful in regulatingexpression of the transcript; see, for example, Parizotto et al. (2004)Genes Dev., 18:2237-2242. Recognition sites of miRNAs have beenvalidated in all regions of an mRNA, including the 5′ untranslatedregion, coding region, and 3′ untranslated region, indicating that theposition of the miRNA target site relative to the coding sequence maynot necessarily affect suppression (see, e.g., Jones-Rhoades and Bartel(2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520,Allen et al. (2004) Nat. Genet., 36:1282-1290, Sunkar and Zhu (2004)Plant Cell, 16:2001-2019). Because miRNAs are important regulatoryelements in eukaryotes, transgenic suppression of miRNAs is useful formanipulating biological pathways and responses. Finally, promoters ofMIR genes can have very specific expression patterns (e.g.,cell-specific, tissue-specific, temporally specific, or inducible), andthus are useful in recombinant constructs to induce such specifictranscription of a DNA sequence to which they are operably linked.Various utilities of miRNAs, their precursors, their recognition sites,and their promoters are described in detail in U.S. Patent ApplicationPublication 2006/0200878 A1, incorporated by reference herein.Non-limiting examples of these utilities include: (1) the expression ofa native miRNA or miRNA precursor sequence to suppress a target gene;(2) the expression of an artificial miRNA or miRNA precursor sequence tosuppress a target gene; (3) expression of a transgene with a miRNArecognition site, where the transgene is suppressed when the maturemiRNA is expressed; (4) expression of a transgene driven by a miRNApromoter.

Designing an artificial miRNA sequence can be as simple as substitutingsequence that is complementary to the intended target for nucleotides inthe miRNA stem region of the miRNA precursor, as demonstrated by Zeng etal. (2002) Mol. Cell, 9:1327-1333. One non-limiting example of a generalmethod for determining nucleotide changes in the native miRNA sequenceto produce the engineered miRNA precursor includes the following steps:(a) Selecting a unique target sequence of at least 18 nucleotidesspecific to the target gene, e.g., by using sequence alignment toolssuch as BLAST (see, for example, Altschul et al. (1990) J. Mol. Biol.,215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402),for example, of both tobacco cDNA and genomic DNA databases, to identifytarget transcript orthologues and any potential matches to unrelatedgenes, thereby avoiding unintentional silencing of non-target sequences;(b) Analyzing the target gene for undesirable sequences (e.g., matchesto sequences from non-target species), and score each potential 19-mersegment for GC content, Reynolds score (see Reynolds et al. (2004)Nature Biotechnol., 22:326-330), and functional asymmetry characterizedby a negative difference in free energy (“.DELTA..DELTA.G” or “ΔΔG”)(see Khvorova et al. (2003) Cell, 115:209-216). Preferably 19-mers areselected that have all or most of the following characteristics: (1) aReynolds score>4, (2) a GC content between about 40% to about 60%, (3) anegative ΔΔG, (4) a terminal adenosine, (5) lack of a consecutive run of4 or more of the same nucleotide; (6) a location near the 3′ terminus ofthe target gene; (7) minimal differences from the miRNA precursortranscript. Positions at every third nucleotide in an siRNA have beenreported to be especially important in influencing RNAi efficacy and analgorithm, “siExplorer” is publicly available atrna.chem.t.u-tokyo.ac.jp/siexplorer.htm (see Katoh and Suzuki (2007)Nucleic Acids Res., 10.1093/nar/gkl1120); (c) Determining the reversecomplement of the selected 19-mers to use in making a modified maturemiRNA. The additional nucleotide at position 20 is preferably matched tothe selected target sequence, and the nucleotide at position 21 ispreferably chosen to either be unpaired to prevent spreading ofsilencing on the target transcript or paired to the target sequence topromote spreading of silencing on the target transcript; and (d)transforming the artificial miRNA into a plant.

In one aspect, an artificial miRNA provided herein is complementary to apolynucleotide having at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto a polynucleotide selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 69, 71, 73, 75, 77, 83-160,186, 188, 190, 196, 198, 223, 225-228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, and fragments thereof. In anotheraspect, an artificial miRNA provided herein is complementary to apolynucleotide encoding a polypeptide having at least 40%, at least 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 70, 72, 74, 76, 78, 161-185, 189, 191, 197, 199, 224, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255. In yetanother aspect, an artificial miRNA provided herein is complementary toa polynucleotide encoding a polypeptide having at least 40%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence similarity to a polypeptide selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 189, 191, 197, 199, 224,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255.

In one aspect, an artificial miRNA provided herein reduces or eliminatesRNA transcription or protein translation of a target gene.

In one aspect, a miRNA or an artificial miRNA provided herein is underthe control of a tissue specific promoter. In another aspect, a miRNA oran artificial miRNA provided herein is under the control of a promoterselected from the group consisting of SEQ ID NOs: 113-118, 148-160, 204,and a fragment thereof. In one aspect, a modified plant provided hereincomprises an artificial miRNA under the control of a heterologouspromoter selected from the group consisting of SEQ ID NOs: 113-118,148-160, 204, and a fragment thereof.

Plant microRNAs regulate their target genes by recognizing and bindingto a near-perfectly complementary sequence (miRNA recognition site) inthe target transcript, followed by cleavage of the transcript by RNaseIII enzymes such as Argonautel. In plants, certain mismatches between agiven miRNA recognition site and the corresponding mature miRNA are nottolerated, particularly mismatched nucleotides at positions 10 and 11 ofthe mature miRNA. Positions within the mature miRNA are given in the 5′to 3′ direction. Perfect complementarity between a given miRNArecognition site and the corresponding mature miRNA is usually requiredat positions 10 and 11 of the mature miRNA. See, for example,Franco-Zorrilla et al. (2007) Nature Genetics, 39:1033-1037; and Axtellet al. (2006) Cell, 127:565-577.

This characteristic of plant miRNAs is exploited to arrive at rules forpredicting a “microRNA decoy sequence”, i.e., a sequence that can berecognized and bound by an endogenous mature miRNA resulting inbase-pairing between the miRNA decoy sequence and the endogenous maturemiRNA, thereby forming a cleavage-resistant RNA duplex that is notcleaved because of the presence of mismatches between the miRNA decoysequence and the mature miRNA. Mismatches include canonical mismatches(e. g., G-A, C-U, C-A) as well as G::U wobble pairs and indels(nucleotide insertions or deletions). In general, these rules define (1)mismatches that are required, and (2) mismatches that are permitted butnot required.

Required mismatches include: (a) at least 1 mismatch between the miRNAdecoy sequence and the endogenous mature miRNA at positions 9, 10, or 11of the endogenous mature miRNA, or alternatively, (b) 1, 2, 3, 4, or 5insertions (i. e., extra nucleotides) at a position in the miRNA decoysequence corresponding to positions 9, 10, or 11 of the endogenousmature miRNA.

Mismatches that are permitted, but not required, include: (a) 0,1, or 2mismatches between the miRNA decoy sequence and the endogenous maturemiRNA at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of the endogenousmature miRNA, and (b) 0, 1, 2, or 3 mismatches between the miRNA decoysequence and the endogenous mature miRNA at positions 12 through thelast position of the endogenous mature miRNA (i. e., at position 21 of a21-nucleotide mature miRNA), where each of the mismatches at positions12 through the last position of the endogenous mature miRNA is adjacentto at least one complementary base-pair (i. e., so that there is notmore than 2 contiguous mismatches at positions 12 through the lastposition of the endogenous mature miRNA).

A miRNA decoy sequence can be of any length as long as it is recognizedand bound by an endogenous mature miRNA to form a cleavage-resistant RNAduplex. In one aspect, a miRNA decoy sequence includes between about 18to about 36 nucleotides. In another aspect, a microRNA decoy providedherein is a small RNA molecule of at least 15, at least 16, at least 17,at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, or more than 30 nucleotides that iscapable of binding to a mature miRNA. See, for example, WO 2008/133643,which is herein incorporated by reference in its entirety.

In one aspect, an endogenous miRNA is regulated by a miRNA decoyprovided herein. A microRNA decoy provided herein is capable ofpreventing a complementary mature miRNA from binding its native targetgene, thereby increasing expression of the target gene.

In another aspect, a recombinant DNA construct provided herein comprisesat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, or more than 10 miRNAdecoys. In one aspect, a miRNA decoy provided herein is under thecontrol of a regulatory sequence selected from the group consisting ofSEQ ID NOs: 113-118, 148-160, 204, and a fragment thereof.

In another aspect, an endogenous miRNA target is edited with asite-specific nuclease provided herein to mutate at least one miRNAbinding site, thereby rendering the endogenous miRNA target resistant tomiRNA-mediated degradation. As used herein, a “miRNA target” refers to acontiguous stretch of at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, or more than 30nucleotides having at least 75%, at least 80%, at least 85%, at least88%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identity to a mature miRNA. In one aspect, a miRNA target iscapable of being hybridized by a mature miRNA, and then cleaved by amature miRNA/Argonaute RNA-induced silencing complex, under typicalcellular conditions.

Also provided herein is cured tobacco material made from tobacco plantsor plant components provided herein. “Curing” is the aging process thatreduces moisture and brings about the destruction of chlorophyll givingtobacco leaves a golden color and by which starch is converted to sugar.Cured tobacco therefore has a higher reducing sugar content and a lowerstarch content compared to harvested green leaf. In one aspect, tobaccoplants or plant components provided herein can be cured usingconventional means, e.g., flue-cured, barn-cured, fire-cured, air-curedor sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford) for a description of different types of curingmethods. Cured tobacco is usually aged in a wooden drum (e.g., ahogshead) or cardboard cartons in compressed conditions for severalyears (e.g., two to five years), at a moisture content ranging from 10%to about 25%. See, U.S. Pat. Nos. 4,516,590 and 5,372,149. Cured andaged tobacco then can be further processed. Further processing includesconditioning the tobacco under vacuum with or without the introductionof steam at various temperatures, pasteurization, and fermentation.Fermentation typically is characterized by high initial moisturecontent, heat generation, and a 10 to 20% loss of dry weight. See, forexample, U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S.Publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford). Cured, aged, and fermented tobacco can be furtherprocessed (e.g., cut, shredded, expanded, or blended). See, for example,U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. In one aspect, thecured tobacco material of the present disclosure is flue-cured,sun-cured, air-cured, or fire-cured.

Tobacco material obtained from modified tobacco lines, varieties orhybrids of the present disclosure can be used to make tobacco products.As used herein, “tobacco product” is defined as any product made orderived from tobacco that is intended for human use or consumption. Inan aspect, a tobacco product provided herein comprises cured componentsfrom a modified tobacco plant provided herein. In another aspect, atobacco product provided herein comprises cured tobacco leaves from amodified tobacco plant provided herein.

Tobacco products provided herein include, without limitation, cigaretteproducts (e.g., cigarettes, bidi cigarettes, kreteks), cigar products(e.g., cigars, cigar wrapping tobacco, cigarillos), pipe tobaccoproducts, products derived from tobacco, tobacco-derived nicotineproducts, smokeless tobacco products (e.g., moist snuff, dry snuff,chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, longcut chewing tobacco, pouched chewing tobacco), films, chewables (e.g.,gum), lozenges, dissolving strips, tabs, tablets, shaped parts, gels,consumable units, insoluble matrices, hollow shapes, reconstitutedtobacco, expanded tobacco, and the like. See, for example, U.S. PatentPublication No. US 2006/0191548.

As used herein, “cigarette” refers a tobacco product having a “rod” and“filler”. The cigarette “rod” includes the cigarette paper, filter, plugwrap (used to contain filtration materials), tipping paper that holdsthe cigarette paper (including the filler) to the filter, and all gluesthat hold these components together. The “filler” includes (1) alltobaccos, including but not limited to reconstituted and expandedtobacco, (2) non-tobacco substitutes (including but not limited toherbs, non-tobacco plant materials and other spices that may accompanytobaccos rolled within the cigarette paper), (3) casings, (4)flavorings, and (5) all other additives (that are mixed into tobaccosand substitutes and rolled into the cigarette).

In one aspect, this disclosure provides nicotine derived from and amethod of producing nicotine from a modified tobacco plant providedherein for use in a product.

In one aspect, a method provided herein comprises preparing a tobaccoproduct using a cured tobacco leaf from a modified tobacco plantprovided herein.

As used herein, “reconstituted tobacco” refers to a part of tobaccofiller made from tobacco dust and other tobacco scrap material,processed into sheet form and cut into strips to resemble tobacco. Inaddition to the cost savings, reconstituted tobacco is very importantfor its contribution to cigarette taste from processing flavordevelopment using reactions between ammonia and sugars.

As used herein, “expanded tobacco” refers to a part of tobacco fillerwhich is processed through expansion of suitable gases so that thetobacco is “puffed” resulting in reduced density and greater fillingcapacity. It reduces the weight of tobacco used in cigarettes.

Tobacco products derived from plants of the present disclosure alsoinclude cigarettes and other smoking articles, particularly thosesmoking articles including filter elements, where the rod of smokeablematerial includes cured tobacco within a tobacco blend. In an aspect, atobacco product of the present disclosure is selected from the groupconsisting of a cigarillo, a non-ventilated recess filter cigarette, avented recess filter cigarette, a bidi cigarette, a cigar, snuff, pipetobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaftobacco, hookah tobacco, shredded tobacco, and cut tobacco. In anotheraspect, a tobacco product of the present disclosure is a smokelesstobacco product. Smokeless tobacco products are not combusted andinclude, but not limited to, chewing tobacco, moist smokeless tobacco,snus, and dry snuff. Chewing tobacco is coarsely divided tobacco leafthat is typically packaged in a large pouch-like package and used in aplug or twist. Moist smokeless tobacco is a moist, more finely dividedtobacco that is provided in loose form or in pouch form and is typicallypackaged in round cans and used as a pinch or in a pouch placed betweenan adult tobacco consumer's cheek and gum. Snus is a heat treatedsmokeless tobacco. Dry snuff is finely ground tobacco that is placed inthe mouth or used nasally. In a further aspect, a tobacco product of thepresent disclosure is selected from the group consisting of loose leafchewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. Inyet another aspect, a tobacco product of the present disclosure isselected from the group consisting of an electronically heatedcigarette, an e-cigarette, an electronic vaporing device.

The present disclosure further provides a method manufacturing a tobaccoproduct comprising tobacco material from tobacco plants provided herein.In one aspect, methods provided herein comprise conditioning agedtobacco material made from tobacco plants provided herein to increaseits moisture content from between about 12.5% and about 13.5% to about21%, blending the conditioned tobacco material to produce a desirableblend. In one aspect, the method of manufacturing a tobacco productprovided herein further comprises casing or flavoring the blend.Generally, during the casing process, casing or sauce materials areadded to blends to enhance their quality by balancing the chemicalcomposition and to develop certain desired flavor characteristics.Further details for the casing process can be found in TobaccoProduction, Chemistry and Technology, Edited by L. Davis and M. Nielsen,Blackwell Science, 1999.

Tobacco material provided herein can be also processed using methodsincluding, but not limited to, heat treatment (e.g., cooking, toasting),flavoring, enzyme treatment, expansion and/or curing. Both fermented andnon-fermented tobaccos can be processed using these techniques. Examplesof suitable processed tobaccos include dark air-cured, dark fire cured,burley, flue cured, and cigar filler or wrapper, as well as the productsfrom the whole leaf stemming operation. In one aspect, tobacco fibersinclude up to 70% dark tobacco on a fresh weight basis. For example,tobacco can be conditioned by heating, sweating and/or pasteurizingsteps as described in U.S. Publication Nos. 2004/0118422 or2005/0178398.

Tobacco material provided herein can be subject to fermentation.Fermenting typically is characterized by high initial moisture content,heat generation, and a 10 to 20% loss of dry weight. See, e.g., U.S.Pat. Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition tomodifying the aroma of the leaf, fermentation can change either or boththe color and texture of a leaf. Also during the fermentation process,evolution gases can be produced, oxygen can be taken up, the pH canchange, and the amount of water retained can change. See, for example,U.S. Publication No. 2005/0178398 and Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford). Cured, or cured and fermented tobacco can befurther processed (e.g., cut, expanded, blended, milled or comminuted)prior to incorporation into the oral product. The tobacco, in somecases, is long cut fermented cured moist tobacco having an ovenvolatiles content of between 48 and 50 weight percent prior to mixingwith a copolymer and, optionally, flavorants and other additives.

In one aspect, tobacco material provided herein can be processed to adesired size. In certain aspects, tobacco fibers can be processed tohave an average fiber size of less than 200 micrometers. In one aspect,tobacco fibers are between 75 and 125 micrometers. In another aspect,tobacco fibers are processed to have a size of 75 micrometers or less.In one aspect, tobacco fibers include long cut tobacco, which can be cutor shredded into widths of about 10 cuts/inch up to about 110 cuts/inchand lengths of about 0.1 inches up to about 1 inch. Double cut tobaccofibers can have a range of particle sizes such that about 70% of thedouble cut tobacco fibers falls between the mesh sizes of −20 mesh and80 mesh.

Tobacco material provided herein can be processed to have a total ovenvolatiles content of about 10% by weight or greater; about 20% by weightor greater; about 40% by weight or greater; about 15% by weight to about25% by weight; about 20% by weight to about 30% by weight; about 30% byweight to about 50% by weight; about 45% by weight to about 65% byweight; or about 50% by weight to about 60% by weight. Those of skill inthe art will appreciate that “moist” tobacco typically refers to tobaccothat has an oven volatiles content of between about 40% by weight andabout 60% by weight (e.g., about 45% by weight to about 55% by weight,or about 50% by weight). As used herein, “oven volatiles” are determinedby calculating the percentage of weight loss for a sample after dryingthe sample in a pre-warmed forced draft oven at 110° C. for 3.25 hours.An oral product can have a different overall oven volatiles content thanthe oven volatiles content of the tobacco fibers used to make the oralproduct. The processing steps described herein can reduce or increasethe oven volatiles content.

In one aspect, tobacco plants, seeds, plant components, plant cells, andplant genomes provided herein are from a tobacco type selected from thegroup consisting of flue-cured tobacco, sun-cured tobacco, air-curedtobacco, dark air-cured tobacco, and dark fire-cured tobacco. In anotheraspect, tobacco plants, seeds, plant components, plant cells, and plantgenomes provided herein are from a tobacco type selected from the groupconsisting of Burley tobacco, Maryland tobacco, bright tobacco, Virginiatobacco, Oriental tobacco, Turkish tobacco, and Galpão tobacco. In oneaspect, a tobacco plants or seed provided herein is a hybrid plants orseed. As used herein, a “hybrid” is created by crossing two plants fromdifferent varieties or species, such that the progeny comprises geneticmaterial from each parent. Skilled artisans recognize that higher orderhybrids can be generated as well. For example, a first hybrid can bemade by crossing Variety C with Variety D to create a C×D hybrid, and asecond hybrid can be made by crossing Variety E with Variety F to createan E×F hybrid. The first and second hybrids can be further crossed tocreate the higher order hybrid (C×D)×(E×F) comprising geneticinformation from all four parent varieties.

Flue-cured tobaccos (also called Virginia of bright tobaccos) amount toapproximately 40% of world tobacco production. Flue-cured tobaccos areoften also referred to as “bright tobacco” because of the golden-yellowto deep-orange color it reaches during curing. Flue-cured tobaccos havea light, bright aroma and taste. Flue-cured tobaccos are generally highin sugar and low in oils. Major flue-cured tobacco growing countries areArgentina, Brazil, China, India, Tanzania and the U.S. In one aspect,modified tobacco plants or seeds provided herein are in a flue-curedtobacco background selected from the group consisting of CC 13, CC 27,CC 33, CC35, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471,NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110,SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and anyvariety essentially derived from any one of the foregoing varieties. Inanother aspect, modified tobacco plants or seeds provided herein are ina flue-cured tobacco background selected from the group consisting ofCoker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF,NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713,Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51,Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108,Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va116, Va 182, and any variety essentially derived from any one of theforegoing varieties. See WO 2004/041006 A1. In further aspects, modifiedtobacco plants, seeds, hybrids, varieties, or lines provided herein arein any flue cured background selected from the group consisting of K326,K346, and NC196.

Air-cured tobaccos include Burley, Maryland, and dark tobaccos. Thecommon factor is that curing is primarily without artificial sources ofheat and humidity. Burley tobaccos are light to dark brown in color,high in oil, and low in sugar. Burley tobaccos are air-cured in barns.Major Burley growing countries are Argentina, Brazil, Italy, Malawi, andthe U.S. Maryland tobaccos are extremely fluffy, have good burningproperties, low nicotine and a neutral aroma. Major Maryland growingcountries include the U.S. and Italy. In one aspect, modified tobaccoplants or seeds provided herein are in a Burley tobacco backgroundselected from the group consisting of Clay 402, Clay 403, Clay 502, Ky14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, HB4488PLC, PD 7319LC,Bu 21×Ky 10, HBO4P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561 andVa 509. In further aspects, modified tobacco plants, seeds, hybrids,varieties, or lines provided herein are in any Burley backgroundselected from the group consisting of TN 90, KT 209, KT 206, KT212, andHB 4488. In another aspect, modified tobacco plants or seeds providedherein are in a Maryland tobacco background selected from the groupconsisting of Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341.

Dark air-cured tobaccos are distinguished from other types primarily byits curing process which gives dark air-cured tobacco its medium- todark-brown color and distinct aroma. Dark air-cured tobaccos are mainlyused in the production of chewing tobacco and snuff. In one aspect,modified tobacco plants or seeds provided herein are in a dark air-curedtobacco background selected from the group consisting of Sumatra, Jatim,Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured,and Paraguan Passado.

Dark fire-cured tobaccos are generally cured with low-burning wood fireson the floors of closed curing barns. Dark fire-cured tobaccos are usedfor making pipe blends, cigarettes, chewing tobacco, snuff andstrong-tasting cigars. Major growing regions for dark fire-curedtobaccos are Tennessee, Kentucky, and Virginia, USA. In one aspect,modified tobacco plants or seeds provided herein are in a darkfire-cured tobacco background selected from the group consisting ofNarrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VHMadole, Little Crittenden, Green Wood, Little Wood, Small Stalk BlackMammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TND950, VA 309, and VA 359.

Oriental tobaccos are also referred to as Greek, aroma and Turkishtobaccos due to the fact that they are typically grown in easternMediterranean regions such as Turkey, Greece, Bulgaria, Macedonia,Syria, Lebanon, Italy, and Romania. The small plant and leaf size,characteristic of today's Oriental varieties, as well as its uniquearoma properties are a result of the plant's adaptation to the poor soiland stressful climatic conditions in which it develop over many pastcenturies. In one aspect, modified tobacco plants or seeds providedherein are in an Oriental tobacco background selected from the groupconsisting of Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon,Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman,Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa,Bucak, Bitlis, Balikesir, and any variety essentially derived from anyone of the foregoing varieties.

In one aspect, modified tobacco plants, seeds, hybrids, varieties, orlines provided herein are essentially derived from or in the geneticbackground of BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37,CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911,Galpão, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737, GL 939, GL 973,GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K 358, K394, K399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC 95, NC925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116, VA 119,KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907,KY 907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, malesterile KY 14×L8, Narrow Leaf Madole, MS KY171, Narrow Leaf Madole(phph), MS Narrow Leaf Madole, MS TND950, PD 7302LC, PD 7305LC, PD7309LC, PD 7312LC, PD 7318LC, PD 7319LC, MSTKS 2002, TKF 2002, TKF 6400,TKF 4028, TKF 4024, KT206LC, KT209LC, KT210LC, KT212LC, NC 100, NC 102,NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207,‘Perique’, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227,Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20,Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97,TN97LC, TN D94, TN D950, a TR (Tom Rosson) Madole, VA 309, VA 359, orany commercial tobacco variety according to standard tobacco breedingtechniques known in the art.

All foregoing mentioned specific varieties of dark air-cured, Burley,Maryland, dark fire-cured, or Oriental type are only listed forexemplary purposes. Any additional dark air-cured, Burley, Maryland,dark fire-cured, Oriental varieties are also contemplated in the presentapplication.

Also provided herein are populations of tobacco plants described herein.In one aspect, a population of tobacco plants provided herein has aplanting density of between about 5,000 and about 8000, between about5,000 and about 7,600, between about 5,000 and about 7,200, betweenabout 5,000 and about 6,800, between about 5,000 and about 6,400,between about 5,000 and about 6,000, between about 5,000 and about5,600, between about 5,000 and about 5,200, between about 5,200 andabout 8,000, between about 5,600 and about 8,000, between about 6,000and about 8,000, between about 6,400 and about 8,000, between about6,800 and about 8,000, between about 7,200 and about 8,000, or betweenabout 7,600 and about 8,000 plants per acre. In another aspect, apopulation of tobacco plants provided herein is in a soil type with lowto medium fertility.

Also provided herein are containers of seeds from tobacco plantsdescribed herein. A container of tobacco seeds of the present disclosuremay contain any number, weight, or volume of seeds. For example, acontainer can contain at least, or greater than, about 100, at least, orgreater than, about 200, at least, or greater than, about 300, at least,or greater than, about 400, at least, or greater than, about 500, atleast, or greater than, about 600, at least, or greater than, about 700,at least, or greater than, about 800, at least, or greater than, about900, at least, or greater than, about 1000, at least, or greater than,about 1500, at least, or greater than, about 2000, at least, or greaterthan, about 2500, at least, or greater than, about 3000, at least, orgreater than, about 3500, at least, or greater than, or about 4000 ormore seeds. Alternatively, the container can contain at least, orgreater than, about 1 ounce, at least, or greater than, about 5 ounces,at least, or greater than, about 10 ounces, at least, or greater than,about 1 pound, at least, or greater than, about 2 pounds, at least, orgreater than, about 3 pounds, at least, or greater than, about 4 pounds,at least, or greater than, about 5 pounds or more seeds. Containers oftobacco seeds may be any container available in the art. By way ofnon-limiting example, a container may be a box, a bag, a packet, apouch, a tape roll, a tube, or a bottle.

The present disclosure also provides methods for breeding tobacco lines,cultivars, or varieties comprising reduced or eliminated suckering.Breeding can be carried out via any known procedures. DNAfingerprinting, SNP mapping, haplotype mapping or similar technologiesmay be used in a marker-assisted selection (MAS) breeding program totransfer or breed a desirable trait or allele into a tobacco plant. Forexample, a breeder can create segregating populations in an F₂ orbackcross generation using F₁ hybrid plants provided herein or furthercrossing the F₁ hybrid plants with other donor plants with anagronomically desirable genotype. Plants in the F₂ or backcrossgenerations can be screened for a desired agronomic trait or a desirablechemical profile using one of the techniques known in the art or listedherein. Depending on the expected inheritance pattern or the MAStechnology used, self-pollination of selected plants before each cycleof backcrossing to aid identification of the desired individual plantscan be performed. Backcrossing or other breeding procedure can berepeated until the desired phenotype of the recurrent parent isrecovered. In one aspect, a recurrent parent in the present disclosurecan be a flue-cured variety, a Burley variety, a dark air-cured variety,a dark fire-cured variety, or an Oriental variety. In another aspect, arecurrent parent can be a modified tobacco plant, line, or variety.Other breeding techniques can be found, for example, in Wernsman, E. A.,and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In:Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillanPublishing Go., Inc., New York, N.Y., incorporated herein by referencein their entirety.

Results of a plant breeding program using modified tobacco plantsdescribed herein includes useful lines, cultivars, varieties, progeny,inbreds, and hybrids of the present disclosure. As used herein, the term“variety” refers to a population of plants that share constantcharacteristics which separate them from other plants of the samespecies. A variety is often, although not always, sold commercially.While possessing one or more distinctive traits, a variety is furthercharacterized by a very small overall variation between individualswithin that variety. A “pure line” variety may be created by severalgenerations of self-pollination and selection, or vegetative propagationfrom a single parent using tissue or cell culture techniques. A varietycan be essentially derived from another line or variety. As defined bythe International Convention for the Protection of New Varieties ofPlants (Dec. 2, 1961, as revised at Geneva on Nov. 10, 1972; on Oct. 23,1978; and on Mar. 19, 1991), a variety is “essentially derived” from aninitial variety if: a) it is predominantly derived from the initialvariety, or from a variety that is predominantly derived from theinitial variety, while retaining the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of the initial variety; b) it is clearly distinguishable fromthe initial variety; and c) except for the differences which result fromthe act of derivation, it conforms to the initial variety in theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety. Essentiallyderived varieties can be obtained, for example, by the selection of anatural or induced mutant, a somaclonal variant, a variant individualfrom plants of the initial variety, backcrossing, or transformation. Afirst tobacco variety and a second tobacco variety from which the firstvariety is essentially derived, are considered as having essentiallyidentical genetic background. A “line” as distinguished from a varietymost often denotes a group of plants used non-commercially, for examplein plant research. A line typically displays little overall variationbetween individuals for one or more traits of interest, although theremay be some variation between individuals for other traits.

In one aspect, the present disclosure provides a method of producing atobacco plant comprising crossing at least one tobacco plant of a firsttobacco variety with at least one tobacco plant of a second tobaccovariety, where the at least one tobacco plant of the first tobaccovariety exhibits no or reduced topping-induced suckering compared to acontrol tobacco plant of the same variety grown under comparableconditions; and selecting for progeny tobacco plants that exhibit no orreduced topping-induced suckering compared to a control tobacco plant ofthe same cross grown under comparable conditions. In one aspect, a firsttobacco variety provided herein comprises modified tobacco plants. Inanother aspect, a second tobacco variety provided herein comprisesmodified tobacco plants. In one aspect, a first or second tobaccovariety is male sterile. In another aspect, a first or second tobaccovariety is cytoplasmically male sterile. In another aspect, a first orsecond tobacco variety is female sterile. In one aspect, a first orsecond tobacco variety is an elite variety. In another aspect, a firstor second tobacco variety is a hybrid.

In one aspect, the present disclosure provides a method of introgressingone or more transgenes into a tobacco variety, the method comprising:(a) crossing a first tobacco variety comprising one or more transgeneswith a second tobacco variety without the one or more transgenes toproduce one or more progeny tobacco plants; (b) genotyping the one ormore progeny tobacco plants for the one or more transgenes; and (c)selecting a progeny tobacco plant comprising the one or more transgenes.In another aspect, these methods further comprise backcrossing theselected progeny tobacco plant with the second tobacco variety. Infurther aspects, these methods further comprise: (d) crossing theselected progeny plant with itself or with the second tobacco variety toproduce one or more further progeny tobacco plants; and (e) selecting afurther progeny tobacco plant comprising the one or more transgenes. Inone aspect, the second tobacco variety is an elite variety.

In one aspect, the present disclosure provides a method of introgressingone or more mutations into a tobacco variety, the method comprising: (a)crossing a first tobacco variety comprising one or more mutations with asecond tobacco variety without the one or more mutations to produce oneor more progeny tobacco plants; (b) genotyping the one or more progenytobacco plants for the one or more mutations; and (c) selecting aprogeny tobacco plant comprising the one or more mutations. In anotheraspect, these methods further comprise backcrossing the selected progenytobacco plant with the second tobacco variety. In further aspects, thesemethods further comprise: (d) crossing the selected progeny plant withitself or with the second tobacco variety to produce one or more furtherprogeny tobacco plants; and (e) selecting a further progeny tobaccoplant comprising the one or more mutations. In one aspect, the secondtobacco variety is an elite variety.

In one aspect, the present disclosure provides a method of growing apopulation of modified tobacco plants comprising no or reducedsuckering, where the method comprises planting a population of tobaccoseeds comprising one or more mutations, one or more transgenes, or both,where the one or more modified tobacco plants exhibit no or reducedsuckering compared to control tobacco plants of the same variety whengrown under comparable conditions.

In one aspect, the present disclosure provides a method of growing amodified tobacco plant comprising planting a modified tobacco seedcomprising a heterologous promoter that is operably linked to apolynucleotide encoding a polypeptide having at least 40%, at least 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193,195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,255; and growing the modified tobacco plant from the seed. In anotheraspect, this disclosure provides a method of growing a modified tobaccoplant comprising planting a modified tobacco seed comprising arecombinant DNA construct comprising a heterologous promoter that isfunctional in an L1 layer, an L2 layer, an L3 region, a rib zone, acentral zone, a peripheral zone, or a combination thereof, and isoperably linked to a polynucleotide that encodes a non-coding RNAmolecule, where the non-coding RNA molecule is capable of binding to anRNA encoding a polypeptide having at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a polypeptide selected from the group consisting ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70,72, 74, 76, 78, 161-185, 187, 189, 191, 197, 199, 224, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, and where thenon-coding RNA molecule suppresses the expression of the polypeptide;and growing the modified tobacco plant the the seed. In an aspect,growing comprises germinating a seed. In another aspect, growingcomprises placing a seedling in soil, agar, agar-based media, or ahydroponics system. In another aspect, growing comprises providing aseed or plant with water, light (e.g., artificial light, sunlight),fertilizer, a rooting media, or a combination thereof. In an aspect,growing can take place indoors (e.g., a greenhouse) or outdoors (e.g., afield). In one aspect, growing comprises placing a seed or a plant in acontainer.

In one aspect, this disclosure provides a method for manufacturing amodified seed, comprising introducing a recombinant DNA constructprovided herein into a plant cell; screening a population of plant cellsfor the recombinant DNA construct; selecting one or more plant cellsfrom the population; generating one or more modified plants from the oneor more plant cells; and collecting one or more modified seeds from theone or more modified plants.

As used herein, “locus” is a chromosome region where a polymorphicnucleic acid, trait determinant, gene, or marker is located. The loci ofthis disclosure comprise one or more polymorphisms in a population;e.g., alternative alleles are present in some individuals. As usedherein, “allele” refers to an alternative nucleic acid sequence at aparticular locus. The length of an allele can be as small as 1nucleotide base, but is typically larger. For example, a first allelecan occur on one chromosome, while a second allele occurs on a secondhomologous chromosome, e.g., as occurs for different chromosomes of aheterozygous individual, or between different homozygous or heterozygousindividuals in a population. As used herein, a chromosome in a diploidplant is “hemizygous” when only one copy of a locus is present. Forexample, an inserted transgene is hemizygous when it only inserts intoone sister chromosome (i.e., the second sister chromosome does notcontain the inserted transgene).

In one aspect, a modified plant, seed, plant component, plant cell, orplant genome is homozygous for a transgene provided herein. In anotheraspect, a modified plant, seed, plant component, plant cell, or plantgenome is heterozygous for a transgene provided herein. In one aspect, amodified plant, seed, plant component, plant cell, or plant genome ishemizygous for a transgene provided herein. In one aspect, a modifiedplant, seed, plant component, plant cell, or plant genome is homozygousfor a mutation provided herein. In another aspect, a modified plant,seed, plant component, plant cell, or plant genome is heterozygous for amutation provided herein. In one aspect, a modified plant, seed, plantcomponent, plant cell, or plant genome is hemizygous for a mutationprovided herein.

As used herein, “introgression” or “introgress” refers to thetransmission of a desired allele of a genetic locus from one geneticbackground to another.

As used herein, “crossed” or “cross” means to produce progeny viafertilization (e.g. cells, seeds or plants) and includes crosses betweendifferent plants (sexual) and self-fertilization (selfing).

As used herein, “backcross” and “backcrossing” refer to the processwhereby a progeny plant is repeatedly crossed back to one of itsparents. In a backcrossing scheme, the “donor” parent refers to theparental plant with the desired gene or locus to be introgressed. The“recipient” parent (used one or more times) or “recurrent” parent (usedtwo or more times) refers to the parental plant into which the gene orlocus is being introgressed. The initial cross gives rise to the F₁generation. The term “BC1” refers to the second use of the recurrentparent, “BC2” refers to the third use of the recurrent parent, and soon. In one aspect, a backcross is performed repeatedly, with a progenyindividual of each successive backcross generation being itselfbackcrossed to the same parental genotype.

As used herein, “elite variety” means any variety that has resulted frombreeding and selection for superior agronomic performance.

As used herein, “selecting” or “selection” in the context of breedingrefer to the act of picking or choosing desired individuals, normallyfrom a population, based on certain pre-determined criteria.

In one aspect, tobacco plants provided herein are hybrid plants. Hybridscan be produced by preventing self-pollination of female parent plants(e.g., seed parents) of a first variety, permitting pollen from maleparent plants of a second variety to fertilize the female parent plants,and allowing F₁ hybrid seeds to form on the female plants.Self-pollination of female plants can be prevented by emasculating theflowers at an early stage of flower development. Alternatively, pollenformation can be prevented on the female parent plants using a form ofmale sterility. For example, male sterility can be produced by malesterility (MS), or transgenic male sterility where a transgene inhibitsmicrosporogenesis and/or pollen formation, or self-incompatibility.Female parent plants containing MS are particularly useful. In aspectsin which the female parent plants are MS, pollen may be harvested frommale fertile plants and applied manually to the stigmas of MS femaleparent plants, and the resulting F₁ seed is harvested. Additionally,female sterile plants can also be used to prevent self-fertilization.

Plants can be used to form single-cross tobacco F₁ hybrids. Pollen froma male parent plant is manually transferred to an emasculated femaleparent plant or a female parent plant that is male sterile to form F₁seed. Alternatively, three-way crosses can be carried out where asingle-cross F₁ hybrid is used as a female parent and is crossed with adifferent male parent. As another alternative, double-cross hybrids canbe created where the F₁ progeny of two different single-crosses arethemselves crossed. Self-incompatibility can be used to particularadvantage to prevent self-pollination of female parents when forming adouble-cross hybrid.

In one aspect, a tobacco variety provided herein is male sterile. Inanother aspect, a tobacco variety provided herein is cytoplasmic malesterile (CMS). Male sterile tobacco plants may be produced by any methodknown in the art. Methods of producing male sterile tobacco aredescribed in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen.Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H.Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp. Inanother aspect, a tobacco variety provided herein is female sterile. Asa non-limiting example, female sterile plants can be made by mutatingthe STIG1 gene. See, for example, Goldman et al. 1994, EMBO Journal13:2976-2984.

As used herein, the term “sequence identity” or “identity” in thecontext of two polynucleotide or polypeptide sequences makes referenceto the residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.”

The use of the term “polynucleotide” is not intended to limit thepresent disclosure to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides and nucleic acidmolecules can comprise ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The polynucleotides of the present disclosure also encompassall forms of sequences including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like.

As used herein, the term “polypeptide” refers to a chain of at least twocovalently linked amino acids.

The present disclosure provides recombinant, purified, isolated, orprocessed nucleic acids and polypeptides. In one aspect, the presentdisclosure provides a nucleic acid molecule comprising at least about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% identity to a sequence selected from the group consistingof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225-228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, and fragments thereof. In one aspect, the present disclosureprovides a nucleic acid molecule comprising at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, at least 27, at least 28, at least 29, at least 30, ormore than 30 contiguous nucleotides identical to a polynucleotideselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83-160,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225-228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, and 254.

In another aspect, the present disclosure provides a polynucleotideencoding a polypeptide comprising at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, about 98%, at least about99%, or about 100% sequence identity to a polypeptide sequence selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187,189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, and 255. In another aspect, the present disclosureprovides a polynucleotide encoding a polypeptide comprising at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or about 100% similarity to apolypeptide sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, and 255.

In one aspect, the present disclosure provides a polypeptide comprisingat least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or about 100%identity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199,201, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255. In oneaspect, the present disclosure provides a polypeptide comprising atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or about 100% similarity toan amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, and 255. In one aspect, thepresent disclosure provides a polypeptide comprising at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 30, at least40, at least 50, or more than 50 contiguous amino acid residuesidentical to an amino acid sequence in a polypeptide selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191,193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, and 255.

In another aspect, the present disclosure provides a biologically activevariant of a protein having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191,193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, and 255. A biologically active variant of a protein of the presentdisclosure may differ from that protein by as few as 1-15 amino acidresidues, as few as 10, as few as 9, as few as 8, as few as 7, as few as6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1amino acid residue. Also provided herein are orthologous genes orproteins of genes or proteins selected from the group consisting of SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201,203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, and 255. “Orthologs”are genes derived from a common ancestral gene and which are found indifferent species as a result of speciation. Orthologs may share atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orgreater sequence identity or similarity at the nucleotide sequenceand/or the amino acid sequence level. Functions of orthologs are oftenhighly conserved among species.

Nucleic acid molecules, polypeptides, or proteins provided herein can beisolated or substantially purified. An “isolated” or “purified” nucleicacid molecule, polypeptide, protein, or biologically active portionthereof, is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide or protein asfound in its naturally occurring environment. For example, an isolatedor purified polynucleotide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. In one aspect, an isolatedpolynucleotide provided herein can contain less than about 5000nucleotides, less than about 4000 nucleotides, less than about 3000nucleotides, less than about 2000 nucleotides, less than about 1000nucleotides, less than about 500 nucleotides, or less than about 100nucleotides of nucleic acid sequence that naturally flank thepolynucleotide in genomic DNA of the cell from which the polynucleotideis derived. In one aspect, an isolated polynucleotide provided hereincan contain 100-5000, 500-5000, 1000-5000, 2000-5000, 3000-5000,4000-5000, 1-500, 1-1000, 1-2000, 1-3000, 1-4000, 1-5000, 100-500,100-1000, 100-2000, 100-3000, or 100-4000 nucleotides of nucleic acidsequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived. In another aspect, anisolated polypeptide provided herein is substantially free of cellularmaterial in preparations having less than about 30%, less than about20%, less than about 10%, less than about 5%, or less than about 1% (bydry weight) of chemical precursors or non-protein-of-interest chemicals.Fragments of the disclosed polynucleotides and polypeptides encodedthereby are also encompassed by the present invention. Fragments of apolynucleotide may encode polypeptide fragments that retain thebiological activity of the native polypeptide. Alternatively, fragmentsof a polynucleotide that are useful as hybridization probes or PCRprimers using methods known in the art generally do not encode fragmentpolypeptides retaining biological activity. Fragments of apolynucleotide provided herein can range from at least about 20nucleotides, about 50 nucleotides, about 70 nucleotides, about 100nucleotides, about 150 nucleotides, about 200 nucleotides, about 250nucleotides, about 300 nucleotides, and up to the full-lengthpolynucleotide encoding the polypeptides of the invention, depending onthe desired outcome.

Nucleic acids can be isolated using techniques routine in the art. Forexample, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides. Polypeptides can bepurified from natural sources (e.g., a biological sample) by knownmethods such as DEAE ion exchange, gel filtration, and hydroxyapatitechromatography. A polypeptide also can be purified, for example, byexpressing a nucleic acid in an expression vector. In addition, apurified polypeptide can be obtained by chemical synthesis. The extentof purity of a polypeptide can be measured using any appropriate method,e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

In one aspect, this disclosure provides methods of detecting recombinantnucleic acids and polypeptides in plant cells. Without being limiting,nucleic acids also can be detected using hybridization. Hybridizationbetween nucleic acids is discussed in detail in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody provided herein can be a polyclonal antibody or a monoclonalantibody. An antibody having specific binding affinity for a polypeptideprovided herein can be generated using methods well known in the art. Anantibody provided herein can be attached to a solid support such as amicrotiter plate using methods known in the art.

Detection (e.g., of an amplification product, of a hybridizationcomplex, of a polypeptide) can be accomplished using detectable labels.The term “label” is intended to encompass the use of direct labels aswell as indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

The following exemplary, non-limiting embodiments are envisioned:

-   -   1. A modified tobacco plant comprising no or reduced suckers        compared to a control tobacco plant of the same variety when        grown under comparable conditions.    -   2. The modified tobacco plant of embodiment 1, wherein said        suckers is topping-induced suckers.    -   3. The modified tobacco plant of embodiment 1, wherein said        modified tobacco plant comprises one or more mutations.    -   4. The modified tobacco plant of embodiment 1, wherein said        modified tobacco plant comprises one or more transgenes.    -   5. The modified tobacco plant of embodiment 3, wherein said one        or more mutations suppress suckers.    -   6. The modified tobacco plant of embodiment 4, wherein said one        or more transgenes suppress suckers.    -   7. The modified tobacco plant of embodiment 3, wherein said one        or more mutations suppress topping-induced suckers.    -   8. The modified tobacco plant of embodiment 4, wherein said one        or more transgenes suppress topping-induced suckers.    -   9. The modified tobacco plant of embodiment 3, wherein said one        or more mutations suppress suckers prior to topping.    -   10. The modified tobacco plant of embodiment 4, wherein said one        or more transgenes suppress suckers prior to topping.    -   11. The modified tobacco plant of embodiment 3, wherein said one        or more mutations are selected from the group consisting of an        insertion, a deletion, an inversion, a substitution, and a        combination thereof.    -   12. The modified tobacco plant of embodiment 4, wherein said one        or more transgenes comprise an axillary meristem-specific        promoter.    -   13. The modified tobacco plant of embodiment 12, wherein said        axillary meristem-specific promoter is functional or        preferentially functional in an L1 layer, an L2 layer, an L3        region, or a combination thereof.    -   14. The modified tobacco plant of embodiment 12, wherein said        axillary meristem-specific promoter is functional or        preferentially functional in a central zone, a peripheral zone,        a rib zone, or a combination thereof of an axillary meristem.    -   15. The modified tobacco plant of embodiment 11, wherein said        one or more mutations is introduced via a system selected from        the group consisting of chemical mutagenesis, irradiation        mutagenesis, transposon mutagenesis, Agrobacterium-mediated        transformation, a meganuclease, a zinc-finger nuclease (ZFN), a        transcription activator-like effector nuclease (TALEN), a        clustered regularly-interspaced short palindromic repeats        (CRISPR)/Cas9 system, a CRISPR/Cpf1 system, and a combination        thereof.    -   16. The modified tobacco plant of embodiment 11, wherein said        one or more mutations are in a gene encoding a polypeptide        having at least 70% sequence identity to a polypeptide selected        from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,        16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,        48, 50, 52, 54, 58, 60, 62, 68, 70, 72, 74, 76, 78, 80, 82,        161-185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 206, 208,        210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233, 235, 237,        239, 241, 243, 245, 247, 249, 251, 253, 255.    -   17. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant has a similar or higher leaf yield        compared to said control tobacco plant when grown under        comparable conditions.    -   18. The modified tobacco plant of embodiment 17, wherein said        higher leaf yield is at least 0.5%, 1%, 2.5%, 5%, 10%, 15%, or        at least 20% higher.    -   19. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant has a similar plant height compared        to said control tobacco plant when grown under comparable        conditions.    -   20. The modified tobacco plant of embodiment 19, wherein said        similar plant height is within 1%, 5%, 10%, 20%, or 25%.    -   21. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant has a similar cured leaf chemistry        profile compared to said control tobacco plant when grown under        comparable conditions.    -   22. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant produces cured leaves that have a        similar or higher USDA grade index value compared to cured        leaves from said control tobacco plant when grown under        comparable conditions.    -   23. The modified tobacco plant of embodiment 1, wherein said        reduced topping-induced suckers comprises fewer total suckers,        smaller suckers, or both when compared to topping-induced        suckers of a control tobacco plant when grown under comparable        conditions.    -   24. The modified tobacco plant of embodiment 23, wherein said        smaller suckers comprise reduced mass, reduced length, or both        when compared to topping-induced suckers of a control tobacco        plant when grown under comparable conditions.    -   25. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant requires reduced management for        controlling suckers compared to a control tobacco plant when        grown under comparable conditions.    -   26. The modified tobacco plant of embodiment 25, wherein said        reduced management comprises reduced manual removal frequency to        control suckers, reduced chemical application frequency to        control suckers, reduced quantities of chemical application to        control suckers, or any combination thereof compared to a        control tobacco plant when grown under comparable conditions.    -   27. The modified tobacco plant of embodiment 26, wherein said        reduced manual removal frequency to control suckers comprises        less than 10%, less than 20%, less than 30%, less than 40%, less        than 50%, less than 60%, less than 70%, less than 75%, less than        80%, less than 85%, less than 90%, or less than 95% as        frequently as a control plant when grown under comparable        conditions.    -   28. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant is homozygous for said one or more        transgenes or said one or more mutations.    -   29. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant is hemizygous for said one or more        transgenes or said one or more mutations.    -   30. The modified tobacco plant of embodiment 3 or 4, wherein        said modified tobacco plant is heterozygous for said one or more        transgenes or said one or more mutations.    -   31. The modified tobacco plant of embodiment 1, wherein said        plant is selected from the group consisting of a flue-cured        variety, a bright variety, a Burley variety, a Virginia variety,        a Maryland variety, a dark variety, an Oriental variety, and a        Turkish variety.    -   32. The modified tobacco plant of embodiment 1, wherein said        tobacco plant is selected from the group consisting a BU 64        plant, a CC 101 plant, a CC 200 plant, a CC 13 plant, a CC 27        plant, a CC 33 plant, a CC 35 plant, a CC 37 plant, a CC 65        plant, a CC 67 plant, a CC 301 plant, a CC 400 plant, a CC 500        plant, CC 600 plant, a CC 700 plant, a CC 800 plant, a CC 900        plant, a CC 1063 plant, a Coker 176 plant, a Coker 319 plant, a        Coker 371 Gold plant, a Coker 48 plant, a CU 263 plant, a DF911        plant, a Galpao plant, a GL 26H plant, a GL 338 plant, a GL 350        plant, a GL 395 plant, a GL 600 plant, a GL 737 plant, a GL 939        plant, a GL 973 plant, a GF 157 plant, a GF 318 plant, an RJR        901 plant, an HB 04P plant, a K 149 plant, a K 326 plant, a K        346 plant, a K 358 plant, a K394 plant, a K 399 plant, a K 730        plant, an NC 196 plant, an NC 37NF plant, an NC 471 plant, an NC        55 plant, an NC 92 plant, an NC2326 plant, an NC 95 plant, an NC        925 plant, a PVH 1118 plant, a PVH 1452 plant, a PVH 2110 plant,        a PVH 2254 plant, a PVH 2275 plant, a VA 116 plant, a VA 119        plant, a KDH 959 plant, a KT 200 plant, a KT204LC plant, a KY 10        plant, a KY 14 plant, a KY 160 plant, a KY 17 plant, a KY 171        plant, a KY 907 plant, a KY 907LC plant, a KTY14×L8 LC plant, a        Little Crittenden plant, a McNair 373 plant, a McNair 944 plant,        a male sterile KY 14×L8 plant, a Narrow Leaf Madole plant, a MS        KY171 plant, a Narrow Leaf Madole (phph) plant, a MS Narrow Leaf        Madole plant, a MS TND950 plant, a PD 7302LC plant, a PD 7305LC        plant, a PD 7309LC plant, a PD 7312LC plant, a PD 7318LC plant,        a PD 7319LC plant, a MSTKS 2002 plant, a TKF 2002 plant, a TKF        6400 plant, a TKF 4028 plant, a TKF 4024 plant, a KT206LC plant,        a KT209LC plant, a KT210LC plant, a KT212LC plant, an NC 100        plant, an NC 102 plant, an NC 2000 plant, an NC 291 plant, an NC        297 plant, an NC 299 plant, an NC 3 plant, an NC 4 plant, an NC        5 plant, an NC 6 plant, an NC7 plant, an NC 606 plant, an NC 71        plant, an NC 72 plant, an NC 810 plant, an NC BH 129 plant, an        NC 2002 plant, a Neal Smith Madole plant, an OXFORD 207 plant, a        ‘Perique’ plant, a PVH03 plant, a PVH09 plant, a PVH19 plant, a        PVH50 plant, a PVH51 plant, an R 610 plant, an R 630 plant, an R        7-11 plant, an R 7-12 plant, an RG 17 plant, an RG 81 plant, an        RG H51 plant, an RGH 4 plant, an RGH 51 plant, an RS 1410 plant,        a Speight 168 plant, a Speight 172 plant, a Speight 179 plant, a        Speight 210 plant, a Speight 220 plant, a Speight 225 plant, a        Speight 227 plant, a Speight 234 plant, a Speight G-28 plant, a        Speight G-70 plant, a Speight H-6 plant, a Speight H20 plant, a        Speight NF3 plant, a TI 1406 plant, a TI 1269 plant, a TN 86        plant, a TN86LC plant, a TN 90 plant, a TN9OLC plant, a TN 97        plant, a TN97LC plant, a TN D94 plant, a TN D950 plant, a TR        (Tom Rosson) Madole plant, a VA 309 plant, and a VA 359 plant.    -   33. The modified tobacco plant of embodiment 1, wherein said        modified tobacco plant is a hybrid.    -   34. The modified tobacco plant of embodiment 1, wherein said        modified tobacco plant is male sterile or cytoplasmically male        sterile (CMS).    -   35. The modified tobacco plant of embodiment 1, wherein said        modified tobacco plant is female sterile.    -   36. A tobacco leaf of the modified tobacco plant of embodiment        1.    -   37. The tobacco leaf of embodiment 35, wherein said tobacco leaf        is a cured tobacco leaf.    -   38. The tobacco leaf of embodiment 36, wherein said cured        tobacco leaf is air-cured, fire-cured, sun-cured, or flue-cured.    -   39. A tobacco product comprising cured tobacco material from the        modified tobacco plant of embodiment 1.    -   40. The tobacco product of embodiment 39, wherein said tobacco        product is selected from the group consisting of a cigarette, a        kretek, a bidi cigarette, a cigar, a cigarillo, a non-ventilated        cigarette, a vented recess filter cigarette, pipe tobacco,        snuff, chewing tobacco, moist smokeless tobacco, fine cut        chewing tobacco, long cut chewing tobacco, pouched chewing        tobacco product, gum, a tablet, a lozenge, and a dissolving        strip.    -   41. A seed giving rise to the modified tobacco plant of        embodiment 1.    -   42. A method comprising preparing a tobacco product using a        cured tobacco leaf from the modified tobacco plant of embodiment        1.    -   43. A modified tobacco plant, wherein said modified tobacco        plant exhibits:        -   a. inhibited or eliminated axillary meristem growth;        -   b. inhibited or eliminated axillary meristem maintenance; or        -   c. a combination thereof        -   compared to a control tobacco plant of the same variety when            grown under comparable conditions.    -   44. A plant or seed comprising a recombinant polynucleotide,        wherein said recombinant polynucleotide comprises:        -   a. a promoter that is functional in an L1 layer, an L2            layer, an L3 region, a rib zone, a central zone, a            peripheral zone, or any combination thereof, which is            operably linked to        -   b. a structural nucleic acid molecule comprising a nucleic            acid sequence, wherein said nucleic acid sequence encodes a            polypeptide having at least 70% sequence identity to a            polypeptide selected from the group consisting of SEQ ID            NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,            32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 58, 60, 62,            68, 70, 72, 74, 76, 78, 80, 82, 161-185, 189, 191, 193, 195,            197, 199, 201, 203, 206, 208, 210, 212, 214, 216, 218, 220,            222, 224, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,            249, 251, 253, 255.    -   45. The plant or seed of embodiment 43, wherein said promoter        comprises a nucleic acid sequence having at least 90% sequence        identity to a polynucleotide selected from the group consisting        of SEQ ID NOs: 113-118, 148-160, 204 and fragments thereof.    -   46. The plant or seed of embodiment 43, wherein said plant or        seed is a tobacco plant or seed.    -   47. A recombinant DNA construct comprising:        -   a. a promoter that is functional in an L1 layer, an L2            layer, an L3 region, a rib zone, a central zone, a            peripheral zone, or a combination thereof; and        -   b. a heterologous and operably linked nucleic acid sequence,            wherein said nucleic acid sequence encodes a non-coding RNA            or a polypeptide.    -   48. The recombinant DNA construct of embodiment 46, wherein said        promoter comprises a nucleic acid sequence having at least 90%        sequence identity to a polynucleotide selected from the group        consisting of SEQ ID NOs: 113-118, 148-160, 204 and fragments        thereof.    -   49. A method of reducing or eliminating topping-induced suckers        in a tobacco plant, said method comprising transforming a        tobacco plant with a recombinant DNA construct comprising a        promoter functional in an L1 layer, an L2 layer, an L3 region, a        rib zone, a central zone, a peripheral zone, or a combination        thereof.    -   50. The method of embodiment 48, wherein said promoter comprises        a nucleic acid sequence having at least 90% sequence identity to        a polynucleotide selected from the group consisting of SEQ ID        NOs: 113-118, 148-160, 204 and fragments thereof.    -   51. A method comprising transforming a tobacco plant with a        recombinant DNA construct comprising a heterologous promoter        that is functional in an L1 layer, an L2 layer, an L3 region, a        rib zone, a central zone, a peripheral zone, or a combination        thereof, and is operably linked to a polynucleotide that is        transcribed into an RNA molecule that suppresses the level of an        endogenous gene, and wherein said endogenous gene promotes or is        required for axillary meristem growth, axillary meristem        maintenance, or both.    -   52. A method for producing a tobacco plant comprising:        -   a. crossing at least one tobacco plant of a first tobacco            variety with at least one tobacco plant of a second tobacco            variety, wherein said at least one tobacco plant of said            first tobacco variety exhibits no or reduced topping-induced            suckers compared to a control tobacco plant of the same            variety grown under comparable conditions; and        -   b. selecting for progeny tobacco plants that exhibit no or            reduced topping-induced suckers compared to a control            tobacco plant of the same cross grown under comparable            conditions.    -   53. A tobacco plant, or part thereof, comprising a heterologous        promoter operably linked to a polynucleotide encoding a        polypeptide having at least 70% sequence identity to a        polypeptide selected from the group consisting of SEQ ID NOs: 2,        4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,        38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 68, 70, 72,        74, 76, 78, 80, 82, 161-185, 189, 191, 193, 195, 197, 199, 201,        203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231,        233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255.    -   54. The tobacco plant, or part thereof, of embodiment 52,        wherein said promoter comprises a nucleic acid molecule having        at least 90% sequence identity to a polynucleotide selected from        the group consisting of SEQ ID NOs: 113-118, 148-160, 204 and        fragments thereof.    -   55. The tobacco plant, or part thereof, of embodiment 52,        wherein said polynucleotide has at least 90% sequence identity        to a polynucleotide selected from the group consisting of SEQ ID        NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,        33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 67,        69, 71, 73, 75, 77, 79, 81, 83-160, 186, 188, 190, 192, 194,        196, 198, 200, 202, 204, 205, 207, 209, 211, 213, 215, 217, 219,        221, 223, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,        250, 252, and 254.    -   56. The tobacco plant, or part thereof, of embodiment 52,        wherein said polypeptide comprises at least 15 contiguous amino        acid residues identical to said polypeptide selected from the        group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,        20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,        52, 54, 56, 58, 60, 62, 68, 70, 72, 74, 76, 78, 80, 82, 161-185,        189, 191, 193, 195, 197, 199, 201, 203, 206, 208, 210, 212, 214,        216, 218, 220, 222, 224, 229, 231, 233, 235, 237, 239, 241, 243,        245, 247, 249, 251, 253, 255.    -   57. A recombinant DNA construct comprising a heterologous        promoter operably linked to a polynucleotide encoding a        polypeptide having at least 70% sequence identity to a        polypeptide selected from the group consisting of SEQ ID NOs: 2,        4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,        38, 40, 42, 44, 46, 48, 50, 52, 54, 58, 60, 62, 68, 70, 72, 74,        76, 78, 80, 82, 161-185, 189, 191, 193, 195, 197, 199, 201, 203,        206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,        235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255.    -   58. A method of growing a modified tobacco plant comprising        planting a modified tobacco seed comprising a heterologous        promoter that is operably linked to a polynucleotide encoding a        polypeptide having at least 70% sequence identity to a        polypeptide selected from the group consisting of SEQ ID NOs: 2,        4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,        38, 40, 42, 44, 46, 48, 50, 52, 54, 58, 60, 62, 68, 70, 72, 74,        76, 78, 80, 82, 161-185, 189, 191, 193, 195, 197, 199, 201, 203,        206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231, 233,        235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255; and        growing said modified tobacco plant from said seed.    -   59. A method for controlling topping-induced suckers in a plant        comprising transforming said plant with a recombinant DNA        construct, wherein said recombinant DNA construct comprises a        promoter that is operably linked to a polynucleotide encoding a        polypeptide having at least 70% sequence identity to a        polypeptide selected from the group consisting of SEQ ID NOs: 2,        4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,        38, 40, 42, 44, 46, 48, 50, 52, 54, 58, 60, 62, 68, 70, 72, 74,        76, 78, 80, 82, 161-185, 187, 189, 191, 193, 195, 197, 199, 201,        203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 229, 231,        233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255.    -   60. A tobacco plant, or part thereof, comprising a heterologous        promoter operably linked to a polynucleotide that encodes a        non-coding RNA molecule, wherein said non-coding RNA molecule is        capable of binding to an RNA encoding a polypeptide having at        least 70% sequence identity to a polypeptide selected from the        group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,        20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,        52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185,        187, 189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241,        243, 245, 247, 249, 251, 253, 255, and wherein said non-coding        RNA molecule suppresses the expression of said polypeptide.    -   61. The tobacco plant, or part thereof, of embodiment 59,        wherein said promoter comprises a nucleic acid sequence having        at least 90% sequence identity to a polynucleotide selected from        the group consisting of SEQ ID NOs: 113-118, 148-160, 204 and        fragments thereof.    -   62. The tobacco plant, or part thereof, of embodiment 59,        wherein said polynucleotide has at least 90% sequence identity        to a polynucleotide selected from the group consisting of SEQ ID        NOs: 83-101.    -   63. The tobacco plant, or part thereof, of embodiment 59,        wherein said polynucleotide comprises at least 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 contiguous        nucleotides identical to a polynucleotide selected from the        group consisting of SEQ ID NOs: 83-101.    -   64. A recombinant DNA construct comprising a heterologous        axillary meristem-specific promoter operably linked to a        polynucleotide that encodes a non-coding RNA molecule, wherein        said non-coding RNA molecule is capable of binding to an RNA        encoding a polypeptide having at least 70% sequence identity to        a polypeptide selected from the group consisting of SEQ ID NOs:        2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,        36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,        70, 72, 74, 76, 78, 161-185, 187, 189, 191, 197, 199, 224, 229,        231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,        and wherein said non-coding RNA molecule suppresses the        expression of said polypeptide.    -   65. A method of growing a modified tobacco plant comprising        planting a modified tobacco seed comprising a recombinant DNA        construct comprising a heterologous promoter that is functional        in an L1 layer, an L2 layer, an L3 region, a rib zone, a central        zone, a peripheral zone, or a combination thereof, and is        operably linked to a polynucleotide that encodes a non-coding        RNA molecule, wherein said non-coding RNA molecule is capable of        binding to an RNA encoding a polypeptide having at least 70%        sequence identity to a polypeptide selected from the group        consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,        22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,        54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 187,        189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241, 243,        245, 247, 249, 251, 253, 255, and wherein said non-coding RNA        molecule suppresses the expression of said polypeptide; and        growing said modified tobacco plant from said seed.    -   66. A method for controlling topping-induced suckers in a plant        comprising transforming said plant with a recombinant DNA        construct, wherein said recombinant DNA construct comprises a        heterologous promoter that is functional in an L1 layer, an L2        layer, an L3 region, a rib zone, a central zone, a peripheral        zone, or a combination thereof, and wherein said promoter is        operably linked to a polynucleotide that encodes a non-coding        RNA molecule, wherein said non-coding RNA molecule is capable of        binding to an RNA encoding a polypeptide having at least 70%        sequence identity to a polypeptide selected from the group        consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,        22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,        54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78, 161-185, 187,        189, 191, 197, 199, 224, 229, 231, 233, 235, 237, 239, 241, 243,        245, 247, 249, 251, 253, 255, and wherein said non-coding RNA        molecule suppresses the expression of said polypeptide.    -   67. A bacterial cell comprising the recombinant DNA construct of        any one of embodiments 46, 56, or 63.    -   68. A plant genome comprising the recombinant DNA construct of        any one of embodiments 46, 56, or 63.    -   69. A method for manufacturing a modified seed, said method        comprising:        -   a. introducing the recombinant DNA construct from any one of            embodiments 46, 56, or 63 into a plant cell;        -   b. screening a population of plant cells for said            recombinant DNA construct;        -   c. selecting one or more plant cells from said population;        -   d. generating one or more modified plants from said one or            more plant cells; and        -   e. collecting one or more modified seeds from said one or            more modified plants.    -   70. A method of producing a modified tobacco plant to reduce or        eliminate suckers, said method comprising introducing one or        more mutations in one or more tobacco genome loci.    -   71. The method of embodiment 69, wherein said one or more        mutations are introduced via a system selected from the group        consisting of chemical mutagenesis, irradiation mutagenesis,        transposon mutagenesis, Agrobacterium-mediated transformation, a        meganuclease, a ZFN, a TALEN, a CRISPR/Cas9 system, a        CRISPR/Cpf1 system, and a combination thereof.    -   72. A recombinant DNA construct comprising:        -   a. a promoter that is functional in an L1 layer, an L2            layer, an L3 region, a rib zone, a central zone, a            peripheral zone, or a combination thereof; and        -   b. a heterologous and operably linked to an artificial            microRNA, wherein said artificial miRNA has at least 70%            identity to an RNA encoding a polypeptide having at least            70% sequence identity to a polypeptide selected from the            group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,            18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,            48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 78,            161-185, 187, 189, 191, 197, 199, 224, 229, 231, 233, 235,            237, 239, 241, 243, 245, 247, 249, 251, 253, 255, and            wherein said artificial microRNA suppresses the expression            of said polypeptide.    -   73. A plant or seed comprising a recombinant polynucleotide,        wherein said recombinant polynucleotide comprises:        -   a. a promoter that is functional in an L1 layer, an L2            layer, an L3 region, a rib zone, a central zone, a            peripheral zone, or any combination thereof, which is            operably linked to        -   b. a structural nucleic acid molecule comprising a nucleic            acid sequence, wherein said nucleic acid sequence encodes an            auxin biosynthesis protein or an auxin transport protein.    -   74. A recombinant DNA construct comprising:        -   a. a promoter that is functional in an L1 layer, an L2            layer, an L3 region, a rib zone, a central zone, a            peripheral zone, or any combination thereof; and        -   b. a heterologous and operably linked nucleic acid sequence,            wherein said nucleic acid sequence encodes an auxin            biosynthesis protein or an auxin transport protein.    -   75. A recombinant DNA construct comprising a heterologous        axillary meristem-specific promoter operably linked to a        polynucleotide that encodes an auxin biosynthesis protein or an        auxin transport protein.    -   76. The recombinant DNA construct of embodiment 74, wherein said        auxin biosynthesis protein or auxin transport protein is encoded        by a polynucleotide encoding a polypeptide having at least 70%        sequence identity to a polypeptide selected from the group        consisting of SEQ ID NOs: 235, 237, 239, 241, 243, 245, 247,        249, 251, 253, and 255.    -   77. A tobacco plant, or part thereof, comprising a heterologous        promoter having at least 90% sequence identity to a        polynucleotide selected from the group consisting of SEQ ID NOs:        113-118, 148-160, 204, and fragments thereof operably linked to        a polynucleotide encoding an auxin biosynthesis protein or an        auxin transport protein.    -   78. The tobacco plant, or part thereof, of embodiment 76,        wherein said auxin biosynthesis protein or auxin transport        protein is encoded by a polynucleotide encoding a polypeptide        having at least 70% sequence identity to a polypeptide selected        from the group consisting of SEQ ID NOs: 235, 237, 239, 241,        243, 245, 247, 249, 251, 253, and 255.    -   79. A method for controlling topping-induced suckers in a plant        comprising transforming said plant with a recombinant DNA        construct, wherein said recombinant DNA construct comprises a        promoter that is operably linked to a polynucleotide encoding an        auxin biosynthesis protein or an auxin transport protein.    -   80. The method of embodiment 78, wherein said auxin biosynthesis        protein or auxin transport protein is encoded by a        polynucleotide encoding a polypeptide having at least 70%        sequence identity to a polypeptide selected from the group        consisting of SEQ ID NOs: 235, 237, 239, 241, 243, 245, 247,        249, 251, 253, and 255.    -   81. The modified tobacco plant of embodiment 23, wherein said        fewer total suckers comprises at least 10%, at least 15%, at        least 20%, at least 25%, at least 30%, at least 35%, at least        40%, at least 45%, at least 50%, at least 60%, at least 70%, at        least 80%, at least 90%, or at least 95% fewer total suckers        compared to an unmodified control tobacco plant grown under        comparable conditions    -   82. The modified tobacco plant of embodiment 24, wherein said        reduced mass comprises at least 10%, at least 15%, at least 20%,        at least 25%, at least 30%, at least 35%, at least 40%, at least        45%, at least 50%, at least 60%, at least 70%, at least 80%, at        least 90%, or at least 95% reduced mass compared to the mass of        suckers of an unmodified control tobacco plant grown under        comparable conditions    -   83. The modified tobacco plant of embodiment 24, wherein said        reduced length comprises at least 10%, at least 15%, at least        20%, at least 25%, at least 30%, at least 35%, at least 40%, at        least 45%, at least 50%, at least 60%, at least 70%, at least        80%, at least 90%, or at least 95% reduced length compared to        the length of suckers of an unmodified control tobacco plant        grown under comparable conditions

Having now generally described the disclosure, the same will be morereadily understood through reference to the following examples that areprovided by way of illustration, and are not intended to be limiting ofthe present disclosure, unless specified.

EXAMPLES Example 1 Identification of Topping-Inducible Genes

RNA samples from 4 week old TN90 tobacco plants are obtained from 10tissue types (axillary buds before topping; axillary buds 2 hours aftertopping; axillary buds 6 hours after topping; axillary buds 24 hoursafter topping; axillary buds 72 hours after topping; roots beforetopping; roots 24 hours after topping; roots 72 hours after topping;young leaf at the time of topping; and shoot apical meristem). Theresulting RNA samples (three independently collected samples for eachtissue type) are used as starting material for Illumina 1×100 bpsequencing.

Illumina reads are mapped and used to identify a list of candidate genesexhibiting high axillary bud expression. Expression of candidate genesis confirmed using RT-PCR. See U.S. patent application Ser. No.14/875,928, filed on Oct. 6, 2015, published on Sep. 29, 2016 as US2016/0281100, which is herein incorporated by reference in its entirety.After confirming candidate genes are differentially expressed inaxillary buds, full-length candidate genes are cloned using genespecific primers designed from predicted full-length cDNA sequences(Table 2A). Normalized Illumina read counts for selected loci areprovided in Table 2B.

TABLE 2A Selected full-length candidate tobacco genes exhibitingdifferential expression in axillary buds Polynucleotide Polypeptide SEQID NO Coding Length Length (DNA/peptide) Sequence (nucleotides) (aminoacids) Annotation 1/2 Full length 987 328 Transcription factor confirmedCYCLOIDEA-like 3/4 Full length 318 105 Flower-specific gamma- confirmedthionin 5/6 Full length 1797 598 Polyphenoloxidase confirmed 7/8 Fulllength 1392 463 UDP-glucose:glucosyltransferase confirmed  9/10 Fulllength 405 134 Tumor-related protein confirmed 11/12 Full length 630 209Hypothetical protein confirmed 13/14 Full length 1143 380 TCP1protein-like gene confirmed 15/16 Full length 915 304 Chlorophyllase-2confirmed 17/18 Full length 1353 450 AP2/ERF domain- confirmedcontaining transcription factor 19/20 Full length 732 243 Putativemiraculin confirmed 186/187 Pseudo gene 2340 87 (E,E)-geranyllinaloolsynthase 21/22 Full length 471 156 Oleosin confirmed 23/24 Full length1437 478 ACC synthase confirmed 25/26 Full length 645 214 LOBdomain-containing confirmed protein 18-like 27/28 Full length 2205 734Vicilin-like confirmed antimicrobial peptides cupin super family 29/30Full length 1302 433 Abscisic acid insensitive confirmed 31/32 Fulllength 1266 421 Seipin-like confirmed 33/34 Full length 597 198Transcription factor confirmed CYCLOIDEA-like 35/36 Full length 1038 345Transcription factor confirmed DICHOTOMA-like 37/38 Full length 1014 337Transcription factor confirmed CYCLOIDEA-like

TABLE 2B Normalized Illumina read counts for selected candidate genes.SEQ ID NO Axillary Axillary Buds After Topping Roots After Topping (DNA/Buds Before 2 6 24 72 Roots Before 24 72 Shoot Apical Young Peptide)Topping hrs hrs hrs hrs Topping hrs hrs Meristem Leaf 1/2 1,072 9981,346 663 652 7 9 11 180 47 3/4 1,387 927 3,527 44,790 23,270 108 90 1288,913 72 5/6 763 1,132 1,852 5,559 2,644 110 156 80 513 7  9/10 2,3422,357 2,992 3,143 2,190 38 28 27 26 103 11/12 47 29 54 18 17 1 0 0 23 113/14 128 131 187 69 54 0 1 1 13 0 15/16 124 308 1,619 337 136 217 143160 88 234 17/18 3 162 186 9 9 22 22 29 6 2 19/20 41 98 334 136 101 1 00 50 0 186/187 1,479 1,486 4,216 16,176 12,228 46 36 33 2,144 839 21/2252 27 81 13 9 2 1 3 5 1 23/24 152 114 135 46 45 2 2 2 1 0 25/26 60 34 2217 13 2 4 1 30 1 29/30 624 583 1,279 300 215 14 9 18 71 9 31/32 176 121253 95 70 7 1 1 69 27 33/34 268 279 410 231 207 1 1 1 22 11 35/36 193241 366 117 123 2 2 2 13 1 37/38 394 353 505 207 204 2 2 1 34 2

Example 2 Development of Modified Plants

An expression vector, p45-2-7 (SEQ ID NO: 112; FIG. 1), is used as abackbone to generate multiple transformation vectors (See Examples 6-10and 13-20). p45-2-7 contains a CsVMV promoter, a NOS terminator, and acassette comprising a kanamycin selection marker (NPT II) operablylinked to an Actin2 promoter and a NOS terminator. Nucleic acid vectorscomprising transgenes of interest are introduced into tobacco leaf discsvia Agrobacterium transformation. See, for example, Mayo et al., 2006,Nat Protoc. 1:1105-11 and Horsch et al., 1985, Science 227:1229-1231.

Narrow Leaf Madole (NLM) tobacco plants are grown in Magenta™ GA-7 boxesand leaf discs are cut and placed into Petri plates. Agrobacteriumtumefaciens cells comprising a transformation vector are collected bycentrifuging a 20 mL cell suspension in a 50 mL centrifuge tube at 3500RPM for 10 minutes. The supernatant is removed and the Agrobacteriumtumefaciens cell pellet is re-suspended in 40 mL liquid re-suspensionmedium. Tobacco leaves, avoiding the midrib, are cut into eight 0.6 cmdiscs with a #15 razor blade and placed upside down in a Petri plate. Athin layer of Murashige & Skoog with B5 vitamins liquid re-suspensionmedium is added to the Petri plate and the leaf discs are pokeduniformly with a fine point needle. About 25 mL of the Agrobacteriumtumefaciens suspension is added to the Petri plate and the leaf discsare incubated in the suspension for 10 minutes.

Leaf discs are transferred to co-cultivation Petri plates (½ MS medium)and discs are placed upside down in contact with filter paper overlaidon the co-cultivation TOM medium (MS medium with 20 g/L sucrose; 1 mg/Lindole-3-acetic acid; and 2.5 mg/L 6-benzyl aminopurine (BAP)). ThePetri plate is sealed with parafilm prior to incubation in dim light(60-80 mE/ms) with 18 hours on, 6 hours off photoperiods at 24 degreesCelsius for three days. After incubation, leaf discs are transferred toregeneration/selection TOM K medium Petri plates (TOM medium plus 300mg/L kanamycin). Leaf discs are sub-cultured bi-weekly to fresh TOM Kmedium in dim light with 18 hours on, 6 hours off photoperiods at 24degrees Celsius until shoots become excisable. Shoots from leaves areremoved with forceps and inserted in MS basal medium with 100 mg/Lkanamycin. Shoots on MS basal medium with 100 mg/L kanamycin areincubated at 24 degrees Celsius with 18 hours on, 6 hours offphotoperiods with high intensity lighting (6080 mE/ms) to inducerooting.

When plantlets containing both shoots and roots grow large enough (e.g.,reach approximately half the height of a Magenta™ GA-7 box), they aretransferred to soil. Established seedlings are transferred to agreenhouse for further analysis and to set seed. Evaluation of suckeringphenotypes is conducted by growing modified plants (T0, T1, T2, or latergenerations) and control plants to layby stage. Control plants areeither NLM plants that have not been transformed or NLM plants that havebeen transformed with an empty p45-2-7 vector. Plants that have reachedlayby stage are manually topped (the shoot apical meristem andsurrounding tissue is removed), and axillary bud growth is evaluated atspecific time points after topping. Observations are typically performedat the time of topping (i.e., 0 hours), 24 hours (i.e., 1 day) aftertopping, 7-8 days after topping (i.e., one week), and/or 14-15 days(i.e., two weeks) after topping. Observations comprise qualitativelyexamining the presence or absence of axillary bud growth and overallplant appearance. Observations also comprise quantitatively measuringthe fresh weight of all axillary buds at a specific time point aftertopping and/or measuring the length of all axillary bud outgrowths at aspecific time point after topping.

Example 3 Identification of Tobacco Genes That Function in SuckerDevelopment

Transformation vectors and modified tobacco plants are generated toover-express full-length coding sequences from tobacco genes (e.g., SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, and 69).

As an illustration, SEQ ID NO: 11 is incorporated into a p45-2-7transformation vector, and modified tobacco plants are generated,according to Example 2. Modified tobacco plants (T0 generation) andcontrol tobacco plants are grown to the layby stage, then plants aretopped to remove the shoot apical meristem according to Example 2.Sucker growth is evaluated at the time of topping and one week aftertopping. Overexpression of SEQ ID NO:11 increases bud outgrowth intobacco, indicating expression of SEQ ID NO: 11 promotes sucker growth(FIG. 2).

Example 4 Expression of Non-Tobacco Origin Genes That Affect TobaccoSucker Growth

Multiple genes have been identified to play a role in sucker growth innon-tobacco species. Transformation vectors and modified tobacco plantsare generated to express non-tobacco origin full-length genes (e.g., SEQID NOs: 55, 67, 79, and 81). SEQ ID NO: 81 (encoding Arabidopsisthaliana BRANCHED1 (BRC1)) is incorporated into a p45-2-7 transformationvector and modified tobacco plants are generated according to Example 2.In Arabidopsis, BRC1 is expressed in developing buds, where it functionsto arrest bud development. See, for example, González-Grandío et al.,2013, Plant Cell 25: 834-850, which is herein incorporated by referencein its entirety.

Modified tobacco plants (T0 generation) and control tobacco plants aregrown to the layby stage, then plants are topped to remove the shootapical meristem according to Example 2. Sucker growth is evaluated atthe time of topping and one week after topping (FIG. 3A). Expression ofSEQ ID NO: 81 in tobacco reduces bud outgrowth. These plants alsoexhibit stunted growth (FIG. 3B).

Example 5 Identification of Native Tobacco Genes That Inhibit SuckerGrowth

Transformation vectors and modified tobacco plants are generated to useRNAi to inhibit endogenous tobacco genes (e.g., SEQ ID NOs: 83-107) andidentify their role in sucker outgrowth.

Three tobacco genes (SEQ ID NOs: 1, 13, and 35) are identified asTCP-family proteins having homology to Arabidopsis BRC1. Transformationvectors and modified tobacco plants are generated according to Example2; resulting modified tobacco plants are phenotypically evaluated aftertopping according to Example 2.

A first transformation vector comprises SEQ ID NO: 83 inserted into ap45-2-7 backbone for constitutive suppression of native SEQ ID NO: 1 viaRNAi, and plants comprising this vector are hereinafter referred to asRNAi_1 plants. RNAi_1 tobacco plants (T0 generation) and control tobaccoplants are grown to the layby stage, then plants are topped to removethe shoot apical meristem. Sucker growth is evaluated at the time oftopping and one week after topping. Bud outgrowth is apparent in RNAi_1plants prior to topping, and bud outgrowth increases after topping (FIG.4). RNAi_1 T1 generation plants continue to show increased bud outgrowth(FIGS. 5A and B) at least two weeks after topping. The fresh weight ofall axillary shoots two weeks after topping in T1 RNAi_1 plants averages˜600 grams; the fresh weight of all axillary shoots two weeks aftertopping of control plants is ˜300 grams (FIG. 5B). These resultsindicate SEQ ID NO: 1 functions to inhibit sucker outgrowth in tobacco.

A second transformation vector comprises SEQ ID NO: 86 inserted into ap45-2-7 backbone. This second transformation vector is designed torepress native SEQ ID NO: 13 via RNAi mechanisms, and plants comprisingthis vector are hereinafter referred to as RNAi_7 plants. RNAi_7 tobaccoplants (T0 generation) and control tobacco plants are grown to the laybystage, then plants are topped to remove the shoot apical meristem.Sucker growth is evaluated at the time of topping and one week aftertopping. Bud outgrowth increases in RNAi_7 plants (FIG. 6). T1generation RNAi_7 plants continue to show increased bud outgrowth (FIG.7) at least two weeks after topping. The fresh weight of all axillaryshoots two weeks after topping in seven Ti RNAi_7 plant lines average˜600 grams, ˜700 grams, ˜400 grams, ˜250 grams, ˜200 grams, and ˜375grams; the fresh weight of all axillary shoots two weeks after toppingof control plants is ˜300 grams (FIG. 7). These results indicate SEQ IDNO: 13 functions to inhibit sucker outgrowth in tobacco.

A third transformation vector comprises SEQ ID NO: 95 inserted into ap45-2-7 backbone. This third transformation vector is designed torepress native SEQ ID NO: 35 via RNAi mechanisms, and plants comprisingthis vector are hereinafter referred to as RNAi_18 plants. RNAi_18plants develop axillary branches at every node prior to topping (FIG.8). These results indicate SEQ ID NO: 35 functions to inhibit suckeroutgrowth in tobacco.

Example 6 Identification of Native Tobacco Genes That Promote SuckerGrowth

Some tobacco genes natively function to promote sucker outgrowth.Inhibiting these genes using RNAi constructs decreases sucker outgrowthand positively identifies the genes as promoters of sucker outgrowth intobacco. Transformation vectors designed to inhibit predicted promotersof sucker outgrowth via RNAi mechanisms are created according to Example2; modified tobacco plants comprising the transformation vectors arecreated and phenotypically evaluated according to Example 2.

A transformation vector is created to comprise SEQ ID NO: 101 in ap45-2-7 backbone, which is homologous to a region of CET2, aCENTRORADIALIS (CEN)-like gene from Tobacco (SEQ ID NOs: 108-110). CETgenes are not expressed in the shoot apical meristem in tobacco,although CEN is required for shoot apical meristem growth in Antirrhinummajus. In tobacco, expression of CEN extends the vegetative phase anddelays flowering. See, for example, Amaya et al., 1999, Plant Cell11:1405-1418, which is herein incorporated by reference in its entirety.Plants comprising a transformation vector comprising SEQ ID NO: 101 arehereinafter referred to as RNAi_NtCET2 plants.

RNAi_NtCET2 plants (T0 generation), and control tobacco plants are grownto the layby stage, then plants are topped to remove the shoot apicalmeristem. Sucker growth is evaluated at the time of topping and one weekafter topping. Bud outgrowth is reduced in RNAi_NtCET2 plants (FIG. 9),indicating that native NtCET2 promotes sucker outgrowth.

Another transformation vector comprises SEQ ID NO: 96, and plantscomprising this vector are hereinafter referred to as RNAi_26 plants.RNAi_26 plants (T0 generation) and control tobacco plants are grown tothe layby stage, then plants are topped to remove the shoot apicalmeristem. Sucker growth is evaluated at the time of topping and one weekafter topping. Bud outgrowth decreases in RNAi_26 plants (FIG. 10),indicating that SEQ ID NO: 49 natively functions to promote suckeroutgrowth.

Example 7 Identification of Axillary Bud-Specific Promoters

Expression of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 67,69, 79, 81, and 83-107 (See Examples 6-8) can be better utilized toreduce or eliminate sucker outgrowth in modified plants if thepolynucleotides are expressed in a tissue-dependent manner (e.g., onlyin the axillary bud). The expression pattern of 28 candidate genes isanalyzed, and promoters of the genes having high expression in axillarybuds, but low expression in other tissues, are selected (Table 3).Expression patterns of the candidate genes are confirmed by real-timePCR analysis. Six axillary meristem-specific promoters (SEQ ID NOs:113-118) are cloned by PCR methods from tobacco TN90 genomic DNA usinggene-specific primers.

Expression patterns of candidate promoters are analyzed bytransformation of tobacco with a chimeric candidatepromoter::beta-glucuronidase (GUS) reporter gene within the same plasmidbackbone (p45-2-7) described in Example 2. The chimeric gene isintroduced via Agrobacterium-mediated transformation into an NLM line.GUS staining is used to identify tissue-specific promoter expressionfollowing the method of Crone et al., 2001, Plant Cell Environ.24:869-874.

Briefly, tissue from young seedlings comprising a candidatepromoter::GUS transformation construct is placed in cold 90% acetone onice. When all samples are harvested, samples are placed at roomtemperature for 20 minutes. Samples are placed back on ice and acetoneis removed from the samples. Next, staining buffer (0.2% Triton X-100;50 mM NaHPO₄, pH7.2; 2 mM potassium ferrocyanide) is added to thesamples. X-Gluc is added to the staining buffer to a final concentrationof 2 mM. Staining buffer is removed from the samples and fresh stainingbuffer with X-Gluc is added. The samples are then infiltrated undervacuum, on ice, for 15 to 20 minutes. The samples are incubated at 37degrees Celsius for 2-18 hours before the staining buffer is removed.Samples are washed through an ethanol series (i.e., 10%, 30%, 50%, 70%,95%) in the dark for 30 minutes per wash. Finally, samples aretransferred into 100% ethanol.

GUS-positive plant tissues are examined with a bright-field microscope(Leica Q500MC; Cambridge, England) at a low magnification andphotographed with a digital camera. Results of experiments using threedifferent promoters (SEQ ID NOs: 113, 116, and 117) are shown in FIGS.11, 12, and 13, respectively. These promoter sequences can be used todrive the expression of a sequence of interest exclusively, orpredominantly, within an axillary bud while limiting expression in therest of the plant.

GUS-positive expression, indicating expression driven by SEQ ID NOs:113, 116, and 117, is concentrated in axillary buds. Thus, SEQ ID NOs:113, 116, and 117 are tissue-specific promoters that are active inaxillary buds, but not in stem or leaf tissue (FIGS. 11, 12, and 13).The expression of GUS under the direction of SEQ ID NOs: 113 (PromoterP1, hereinafter) and 116 (Promoter P11, hereinafter) decreases aftertopping, which coincides with the gene expression pattern that isobserved for the endogenous genes that are normally regulated by thesepromoters (FIGS. 11 and 12). Promoter P1 and Promoter P11 are alsofunctional in the tobacco shoot apical meristem. (FIGS. 11 and 12).

In contrast, SEQ ID NO: 117 (Promoter P15, hereinafter) drives GUSexpression in the axillary meristem prior to topping and for at leastfifteen days after topping, which coincides with the gene expressionpattern that is observed for the endogenous gene that is normallyregulated by this promoter (FIGS. 13A-C). Promoter P15 is alsofunctional in the base of the shoot apical meristem (FIG. 13A).

Additional candidate promoters of topping-inducible, andtissue-specific, promoters to control sucker outgrowth include SEQ IDNOs: 148-160 and 204, which represent an axillary bud-specific thionin5′ upstream regulatory sequence (SEQ ID NO: 148); a tobacco lateralsuppressor 1 (LAS1) 5′ upstream regulatory sequence (SEQ ID NO: 149); aLAS1 3′ downstream regulatory sequence (SEQ ID NO: 150); a LAS2 5′upstream regulatory sequence (SEQ ID NO: 151); a LAS2 3′ downstreamregulatory sequence (SEQ ID NO: 152); a tobacco regulator of axillarymeristems1 (RAX1) 5′ upstream regulatory sequence (SEQ ID NO: 153); aRAX1 3′ downstream regulatory sequence (SEQ ID NO: 154); a RAX2 5′upstream regulatory sequence (SEQ ID NO: 155); a RAX2 3′ downstreamregulatory sequence (SEQ ID NO: 156); a Promoter P15 5′ region (SEQ IDNO: 157); a Promoter P15 3′ downstream region, (SEQ ID NO: 158); a 5′upstream regulatory sequence of a P15 homolog (SEQ ID NO: 159); a 3′downstream regulatory sequence of a P15 homolog (SEQ ID NO: 160); and aregulatory region of a P15 homolog from tomato (Solanum lycopersicum)(SEQ ID NO: 204). The sequences are cloned by PCR methods from NLMgenomic DNA using gene-specific primers. These regulatory sequences aretested for their tissue specificity and developmental regulation asshown for Promoters P1, P11, and P15. The regulatory sequences thatexhibit axillary meristem-specific or -preferential expression are usedfor driving heterologous gene expression and modulating sucker growth.

TABLE 3 Selected clones for promoter analysis Length of SEQ ID NOPromoter 113 2248 114 2800 115 3356 116 3150 117 2964 118 941 148 5000149 5000 150 5000 151 5000 152 5000 153 5000 154 5000 155 5000 156 5000157 5000 158 5000 159 5000 160 5000 204 5000

TABLE 4 Axillary bud-preferred promoter cis-elements Cis-regulatory Cis-regulatory  element Nucleotide  element Name SequenceBud Dormancy Element (BDE) CACGTG Axillary Bud Growth (Up1) GGCCCAWAxillary Bud Growth (Up2) AAACCCTA Sucrose Responsive Element  AATAGAAAA(SURE) Sugar Repressive Element (SRE) TTATCC Bud Activation Element or GGCCCAT TCP Binding Element (BAE)

Example 8 Cellular Specificity of Promoter P1, Promoter P15, andPromoter PAB Thionin

To analyze the expression pattern of Promoter P1 (SEQ ID NO: 113) andPromoter P15 (SEQ ID NO: 117) at the cellular level in meristem regions,vectors are produced comprising a green fluorescent protein (GFP) geneunder the control of either Promoter P1 or Promoter P15 as described inExample 2. The chimeric vectors are introduced into an NLM tobacco plantvia Agrobacterium-mediated transformation. GFP expression is observedusing fluorescence microscopy. Promoter P15 is restricted to tissuewithin the axillary buds (FIG. 14A). Promoter P1 has a slightly broaderexpression pattern (FIG. 14B).

To analyze the expression pattern of a 0.9 kb long Promoter PAB Thionin(pABTh-0.9 kb, SEQ ID NO: 118) at the cellular level in meristemregions, a vector is produced comprising a GUS gene under the control ofPromoter PAB Thionin as described in Example 2. The chimeric vector isintroduced into an NLM tobacco plant via Agrobacterium-mediatedtransformation. GUS expression is observed in axillary bud tissue andmeristem tissue (FIG. 15).

Promoters P1, P15, and PAB Thionin (pABth-5 kb, SEQ ID NO: 148) areanalyzed for similar and/or unique cis-regulatory elements. Sixcis-regulatory elements are identified (Table 4) upstream of thetranscriptional start sites of the genes natively regulated by PromotersP1, P15, and PAB Thionin (FIG. 16). These cis-regulatory elements canhave direct and/or indirect effects towards regulating sucker-specificor meristem-specific expression patterns.

Example 9 Efficacy Testing of Sucker Inhibiting Constructs

After testing of the tissue-specific expression patterns of candidatepromoters using promoter::GUS fusion analysis in transgenic plants,vectors and modified plants are constructed as described in Example 2 toexpress target genes only in axillary buds. Exemplary constructs areshown in Table 5.

TABLE 5 Exemplary constructs for axillary bud- specific expression of atarget gene. Promoter Target Gene Construct SEQ ID NO. SEQ ID NO. 1 11317 2 113 104 3 113 7 4 113 41 5 113 5 6 118 17 7 118 104 8 118 7 9 11841 10 118 5 11 115 17 12 115 104 13 115 7 14 115 41 15 115 5 16 117 1717 117 104 18 117 7 19 117 41 20 117 5

Efficacy testing for the impact of Constructs 1-20 is carried out undergreenhouse and field conditions. Transgenic plants and matched wild typecontrols are grown to layby stage, then topped and phenotypicallyevaluated as described in Example 2. Field efficacy testing alsodetermines the type and extent of sucker control chemical applicationneeded under normal agronomical practices.

Example 10 Regulating Axillary Bud Outgrowth via Overexpressing Genes

Sucker outgrowth can be regulated by modifying the expression of genesand/or genetic pathways that regulate branching. Some genes nativelyfunction to restrict bud outgrowth and are defined by mutants withincreased branching, for example the Arabidopsis BRANCHED1 gene (SEQ IDNO:81) and tobacco homologs (SEQ ID NOs: 1, 13, 35, 37, and 39); and theArabidopsis MORE AXILLARY BRANCHING1 (MAX1) and MAX2 genes (SEQ ID NO:193 and 195) and tobacco homologs (SEQ ID NO: 197 and 199). See, forexample, Stirnberg et al., 2002, Development 129: 1131-1141, which isherein incorporated by reference in its entirety.

Transformation vectors are created to overexpress proteins that restrictsucker outgrowth in tobacco. Separate transformation vectors comprisingone of SEQ ID NOs: 1, 13, 35, 37, 39, and 81 are incorporated intop45-2-7 transformation vectors. Additional transformation vectors arecreated comprising one of SEQ ID NOs: 1, 13, 35, 37, 39, and 81 drivenby the axillary bud-specific Promoter P15 (SEQ ID NO: 117). Modifiedtobacco plants are generated from these transformation vectors accordingto Example 2. Modified tobacco plants (T0 generation) and controltobacco plants are then phenotypically evaluated as described in Example2. The modified tobacco plants exhibit reduced sucker growth compared tocontrol tobacco plants.

Example 11 Regulating Axillary Bud Outgrowth by Suppressing Genes ThatPromote Sucker Growth

Some genes promote axillary meristem development and are defined bymutants with decreased branching. For example, the Arabidopsis LAS gene(SEQ ID NO: 201), and homologs in tobacco (SEQ ID NOs: 71 and 73); andthe Arabidopsis RAX gene (SEQ ID NO: 203), as well as tobacco homologs(SEQ ID NOs: 75 and 77). See, for example, Greb et al., 2003, Genes &Development 17: 1175-1187; and Keller et al., 2006, Plant Cell 18:598-611, both of which are herein incorporated by reference in theirentireties.

Transformation vectors comprising RNAi constructs are designed toinhibit tobacco proteins that promote sucker outgrowth. Separatetransformation vectors comprise one of SEQ ID NOs: 71, 73, 75, and 77,which are incorporated into p45-2-7 transformation vectors. Additionaltransformation vectors are created comprising one of SEQ ID NOs: 71, 73,75, and 77 driven by axillary bud-specific Promoter P15 (SEQ ID NO:117). These vectors are used to generate modified tobacco plantsaccording to Example 2. Modified tobacco plants and control tobaccoplants are then phenotypically evaluated as described in Example 2. Themodified tobacco plants exhibit reduced sucker growth compared tocontrol tobacco plants.

Example 12 Regulating Sucker Growth with RNAi, Artificial miRNAs andGene Overexpression

A transformation vector is created in which Promoter P15 (SEQ ID NO:117) drives the expression of SEQ ID NOs: 81 (BRC1) and 101 (RNAitargeting NtCET2) in a tissue-specific manner. The PromoterP15::BRC1::NtCET2 vector over-expresses BRC1 and inhibits NtCET2 inaxillary buds. The transformation vector and modified tobacco plants aregenerated as described in Example 2.

Modified tobacco plants (T0 generation) having a PromoterP15::BRC1::NtCET2 construct and control tobacco plants are grown to thelayby stage, then plants are topped to remove the shoot apical meristem.Sucker growth is evaluated at the time of topping, 8 days after topping,and 15 days after topping (FIG. 17). Expression of SEQ ID NOs: 81 and101, driven by Promoter P15, eliminates sucker growth in tobacco.

Additional transformation vectors are created in which Promoter P15 (SEQID NO: 117) drives the expression of an artificial miRNA designed toreduce the transcription or translation of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 123-147,186, 188, 190, 196, and 198. The transformation vectors and modifiedtobacco plants are generated as described in Example 2.

Modified tobacco plants (T0 generation) having a PromoterP15::artificial miRNA construct and control tobacco plants are grown tothe layby stage, then plants are topped to remove the shoot apicalmeristem. Modified and control tobacco plants are phenotypicallyevaluated according to Example 2.

Example 13 Regulating Sucker Growth via Modifying Cytokinin Synthesisand Distribution

Removing the shoot apical meristem releases axillary buds from dormancyand promotes sucker outgrowth. Auxin derived from an intact shoot apicalmeristem suppresses sucker outgrowth, whereas cytokinin induced byremoval of the shoot apical meristem promotes sucker outgrowth.

Depletion of cytokinin in axillary bud regions by over-expressing genesinvolved in cytokinin catabolism with an axillary bud specific promoteris used to reduce cytokinin and inhibit axillary meristem outgrowth. Forexample, Arabidopsis cytokinin oxidase (CKX; SEQ ID NO: 55), tobaccoCKXs (SEQ ID NOs: 57 and 59); and a tobacco adenosinephosphate-isopentenyltransferase gene (SEQ ID NO: 61) are tested. Atransformation vector comprising SEQ ID NO: 59 driven by Promoter P15(SEQ ID NO: 117) is created according to Example 2. Modified tobaccoplants are generated using this vector, and then phenotypicallyevaluated, according to Example 2. The modified tobacco plants exhibitreduced sucker growth compared to control tobacco plants (FIG. 18).

Example 14 Regulating Sucker Growth via Inhibition of Axillary MeristemStem Cell Signaling

Shoot meristems comprise stem cells that are continuously replenishedthrough a feedback circuit involving the WUSCHEL (WUS)-CLAVATA (CLV)signaling pathway. See, for example, Yadav et al., 2011, Genes &Development 25:2025-2030, which is herein incorporated by reference inits entirety. Genes from this pathway include, e.g., WUS (SEQ ID NOs: 63and 65); CLV1; CLV2; and CLV3 (SEQ ID NOs: 67 and 69). A transformationvector is created comprising SEQ ID NO: 67 driven by Promoter P15 (SEQID NO: 117), which causes overexpression of CLV3 in axillary buds.Modified tobacco plants are generated using this transformation vector,and phenotypically evaluated, according to Example 2. The modifiedtobacco plants exhibit reduced sucker growth compared to control tobaccoplants.

Additional transformation vectors are created according to Example 2 tocomprise either SEQ ID NO: 63 or 65 driven by Promoter P15 (SEQ ID NO:117), which inhibit WUS via RNAi. Modified tobacco plants are generatedusing these transformation vectors, and then phenotypically evaluated,according to Example 2. The modified tobacco plants exhibit reducedsucker growth compared to control tobacco plants.

Arabidopsis SHOOT MERISTEMLESS (STM) is a KNOX protein that is essentialfor shoot meristem formation and maintenance. See, for example, Long etal., 1996, Nature 379:66-69, which is herein incorporated by referencein its entirety. NTH15 (SEQ ID NO: 189) is a tobacco homolog of STM thatis expressed in tobacco meristems. See, for example, Tanaka-Ueguchi etal., 1998, Plant Journal 15:391-400, which is herein incorporated byreferences in its entirety. A transformation vector is createdcomprising an RNAi construct that targets SEQ ID NO: 188 for inhibition,driven by Promoter P15 (SEQ ID NO: 117).Modified tobacco plants aregenerated using these transformation vectors, and then phenotypicallyevaluated, according to Example 2. The modified tobacco plants exhibitreduced sucker growth compared to control tobacco plants.

Example 15 Screening for Genes to Control Suckers Using Agroinfiltration

Expression of some plant genes (without being limiting, e.g., RNases;proteases; cell cycle genes; transcription factors; kinases; caspases)can elicit a cell death response when expressed at certain times and/orcell types. Identification of such genes is desired, as they can beoperably linked to an axillary bud-preferred or axillary bud-specificpromoter (e.g., Promoter P15/SEQ ID NO: 117) to reduce or eliminateaxillary bud growth and/or development.

Agroinfiltration is used to transiently express a tobacco genes (e.g.,SEQ ID NOs: 201-222, 228, 230, and 232) in tobacco leaves. Plant genesof interest are inserted into a pBIN19 plasmid and transformed intoAgrobacterium tumefaciens cells. The transformed bacteria are grown in aliquid culture, washed, and suspended in a buffer solution. The buffersolution containing the transformed A. tumefaciens cells is injectedinto one or more living tobacco leaves. Plant leaf phenotypes are thenevaluated for presence or absence of cellular death after 5 days. Anempty plasmid is used as a negative control, while a plasmid containinga Barnase gene (SEQ ID NO: 79) is used as a positive control. Plantgenes inducing cell death in tobacco leaves are used for reducingaxillary bud growth and/or development. See, for example, FIG. 21.

Nicotiana thaliana mitogen-activated protein kinase kinase 2 (NtMEK2;SEQ ID NO: 232) has been identified as being capable of inducing a celldeath response in tobacco. The expression of SEQ ID NO: 232 is directedto axillary buds by driving its expression with Promoter P15 (SEQ ID NO:117).

Separate transformation vectors are created according to Example 2 tocomprise one of SEQ ID NOs: 201-222, 228, 230, and 232 driven byPromoter P15 (SEQ ID NO: 117). Modified tobacco plants are thengenerated with the Promoter P15::cell death gene vectors, and thenphenotypically evaluated, according to Example 2. The modified tobaccoplants exhibit reduced sucker growth compared to control tobacco plants.

Example 16 Regulating Sucker Growth Using RNases

The presence of some proteins at elevated levels, in cells where theyare not normally expressed, or in subcellular locations where they arenot normally located, can induce cell death. Axillary bud specificpromoters, such as Promoter P1 (SEQ ID NO: 113) and Promoter P15 (SEQ IDNO: 117) are used to the express heterologous genes that are detrimentalto an axillary bud and ultimately result in its death.

RNA-degrading enzymes, such as the bacterial RNase Barnase (SEQ ID NO:79; and see, e.g., Hartley, 1989, Trends in Biochemical Sciences14:450-454.) are used to induce cellular death in the axillary bud whentheir expression is driven by Promoter P15 (SEQ ID NO: 117). Atransformation vector is created according to Example 2 to comprise SEQID NO: 79 driven by Promoter P15 (SEQ ID NO: 117). Modified tobaccoplants are then generated with the Promoter P15::Barnase vectoraccording to Example 2. Modified tobacco plants (T0 generation) andcontrol tobacco plants are grown to the layby stage, then plants aretopped to remove the shoot apical meristem. Sucker growth is observed atthe time of topping, 24 hours after topping, one week after topping, twoweeks after topping, and three weeks after topping (FIGS. 19 and 20).Expression of SEQ ID NO: 79 driven by Promoter P15 eliminates suckeroutgrowth in tobacco.

Additional transformation vectors similar to Promoter P15::Barnase arealso created using endogenous tobacco RNases such as: RNase Phy3 (SEQ IDNO: 123), RNase H (SEQ ID NO: 124), RNase P (SEQ ID NO: 125), RNase III(SEQ ID NO: 126), and RNase T2 (SEQ ID NOs: 127-136) in place of Barnase(SEQ ID NO: 79). Each of these vectors is used to generate modifiedtobacco plants, and to then phenotypically evaluate the tobacco plants,as described in Example 2. The modified tobacco plants exhibit reducedsucker growth compared to control tobacco plants.

Example 17 Regulating Sucker Growth Using Vacuolar Processing Enzymes

Vacuolar processing enzymes (VPEs) are proteases that function in normalplant growth and development, and are also implicated invacuole-dependent programmed cell death through their caspase-likeactivity. Eight out of 17 VPE proteins found in the tobacco genomecomprise protease domains that contain all the residues required forcaspase-like activity (SEQ ID NOs: 137-143). The expression of SEQ IDNOs: 137-143 is directed to axillary buds by driving their expressionwith Promoter P15 (SEQ ID NO: 117). Expression of proteins encoded bySEQ ID NOs: 137-143 can be further restricted to vacuoles withinaxillary bud cells by including an N-terminal vacuolar sorting signal.

Separate transformation vectors are created according to Example 2 tocomprise one of SEQ ID NOs: 137-143 driven by Promoter P15 (SEQ ID NO:117). Modified tobacco plants are then generated with the PromoterP15::VPE vectors, and then phenotypically evaluated, according toExample 2. The modified tobacco plants exhibit reduced sucker growthcompared to control tobacco plants.

Example 18 Regulating Sucker Growth Using Proteases

Additional plant proteases are expressed in certain tissues to eliminatethe cells responsible for axillary shoot meristem development.Proteolytic enzymes are divided into four groups based on theircatalytic domain: aspartic proteases, cysteine proteases,metalloproteases, and serine proteases. All of these protease familiesare found in tobacco and are used to inhibit the development of axillaryshoot meristems. For example, an aspartic protease (SEQ ID NO: 144), acysteine protease (SEQ ID NO: 145), a metalloprotease (SEQ ID NO: 146),or a serine protease (SEQ ID NO: 147) are expressed with a tissuespecific promoter (e.g., SEQ ID NOs: 113-118, 148-160, and 204).

Separate transformation vectors are created according to Example 2 tocomprise one of SEQ ID NOs: 144-147 driven by Promoter P15 (SEQ ID NO:117). Modified tobacco plants are then generated with a PromoterP15::protease vector, and then phenotypically evaluated, according toExample 2. The modified tobacco plants exhibit reduced sucker growthcompared to control tobacco plants.

Example 19 Genome Editing Using TALEN

Transcription activator-like effector nuclease (TALEN) technology isused to modify commercial tobacco varieties such as TN90, K326 andNarrow Leaf Madole. TALENs enable genetic modification through inductionof a double strand break (DSB) in a DNA target sequence. The ensuing DNAbreak repair by either a non-homologous end joining (NHEJ) or ahomology-directed repair (HDR)-mediated pathway is exploited tointroduce a desired modification (e.g., gene disruption, gene correctionor gene insertion).

PEG-mediated protoplast transformation is used to introduce a TALEN anda donor DNA molecule into a plant cell. Tobacco leaves from 4-8 weeksold tobacco plants from sterile culture are cut into small pieces andtransferred into a petri dish containing filter-sterilized enzymesolution containing 1.0% Cellulase onuzuka R10 and 0.5% Macerozym. Theleaf strips in the petri dish are vacuum infiltrated for 30 minutes inthe dark using a desiccator. After incubation, the digested leaves areresuspended by shaking at 45 R.P.M. for 230 minutes, and then filteredthrough a sterilized 100 μm nylon filter by collecting in a 50 mLcentrifuge tube. The solution is applied to Lymphoprep and separated viacentrifugation at 100×g for 10 minutes. Protoplast bands are collectedusing a pipette, and purified protoplasts are washed with an equalvolume of W5n solution containing NaCl, CaCl₂, KCl, MES, and glucose,prior to additional centrifugation for 5 minutes at 2000 R.P.M.Protoplast pellets are resuspended at 2×10⁵/mL in W5n solution, and lefton ice for 30 minutes. Next, supernatant is removed and protoplastpellets are resuspended in filter-sterilized MMM solution containingmannitol, MgCl₂ and MES.

PEG transfection of tobacco protoplasts is performed according to amethod described by Zhang et al. (2013, Plant Physiology 161:20-27) withsome modifications. A 500 μL aliquot of protoplast suspension istransferred into a 10 mL culture tube and 25 μL (−10 μg) of plasmid DNAis slowly added to the protoplasts suspension. Next, 525 μL PEG solutionis added to the protoplast-DNA solution and mixed by carefully tappingthe tube. Tubes are incubated for 20 minutes, then 2.5 mL W5n solutionis added to stop the reaction. The solution is centrifuged at 100×g for5 minutes, and washed with protoplast culture media. PEG-treatedprotoplasts are resuspended in 1 mL culture media containing 0.1 mg/LNAA and 0.5 mg/L BAP, and mixed with 1 mL low-melting agar to makeprotoplast beads. Protoplast beads are cultured in liquid media, andcalli growing from protoplast beads are transferred onto solid shootingmedia. When shoots are well developed, they are transferred into aMagenta™ GA-7 box for root formation. When root systems are fullydeveloped and shoot growth resumes, plants are transplanted into soil.

Multiple TALEN approaches are used to prevent or reduce sucker growth intobacco. Instead of randomly inserting a gene into a tobacco genomeusing conventional transformation methods, TALEN is used for targetedreplacement of an endogenous coding sequence. In one example, a codingsequence of interest (e.g., SEQ ID NOs: 123-147) can be placed under thecontrol of an axillary bud-specific promoter sequence (e.g., SEQ ID NOs:113-118, 148-160, and 204), and the construct can be used with a TALENto homologously recombine the construct into the endogenous genomicregion controlled by the promoter. A TALEN donor sequence is shown inSEQ ID NO:119, and a TALEN target sequence is shown in SEQ ID NO:120.

A second example places an axillary bud-specific promoter and a codingsequence of interest under the control of a native axillary bud-specificpromoter to provide two doses of promoter control. A construct includinga first promoter (SEQ ID NO:118), a second promoter (SEQ ID NO:113), anda coding sequence (SEQ ID NO:13) is homologously recombined into thegenomic region containing native SEQ ID NO: 118 using TALEN, therebydirecting expression of the coding sequence by both promoters (SEQ IDNO:118 and 113). A TALEN donor sequence is shown in SEQ ID NO:121.

A third example uses TALEN to disrupt a target gene that promotes suckergrowth and/or development. TALEN target sequences are identified fornucleic acid sequences (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 69, 71, 73, 75, 77, 108-110, 123-147, 186, 188,190, 196, 198) and nucleic acid sequences encoding polypeptides (e.g.,SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70,72, 74, 76, 78, 161-185, 187, 189, 191, 197, 199). Gene-specific TALENsare designed and introduced into tobacco cells to cause deletions orinsertions in an endogenous target gene. For example, potential TALENtarget sites in a coding sequence (SEQ ID NO: 11) are identified, andhomologous recombination sites within the coding sequence of the geneare selected. A TALEN target sequence is shown in SEQ ID NO:122 (thetarget sequences are underlined).

Example 20 Additional Methods of Regulating Sucker Growth Using GeneEditing Technologies

Gene editing technologies such as CRISPR/Cas9, CRISPR/Cpf1, zinc-fingernucleases (ZFN), and transcription activator-like effector nucleases(TALENs) are used to replace the coding region of an axillarymeristem-specific gene with a cell death/axillary shoot suppressorsequence. These gene editing technologies are also used to edit orreplace an endogenous promoter sequence to drive its cognate proteinexpression in axillary buds. For example, an endogenous RNase promoteris edited or replaced so the RNase is only expressed in axillary buds,where it can function to reduce sucker outgrowth via the induction ofcell death. Alternatively, the promoter of an axillary meristemregulator gene is mutated (edited) to eliminate regulatory region(s)required for timely expression during sucker activation and/oroutgrowth, which can lead to growth defects and/or death of axillaryshoots. Gene editing technologies are further used to edit or replace ofan endogenous gene that natively functions in axillary buds. Anendogenous gene such as NtCET2 is edited so that it no longer makes afunctional protein, thereby inhibiting sucker outgrowth.

Separate CRISPR/Cas9 or CRISPR/Cpf1 guide RNAs are constructed torecognize and hybridize to the promoter sequence of each one of SEQ IDNOs: 123-147. The engineered guide RNA and a donor polynucleotidecomprising Promoter P15 (SEQ ID NO: 117) are provided to a tobaccoplant, allowing Promoter P15 to replace the endogenous promoter of SEQID NO: 123-147 and restrict expression of endogenous SEQ ID NOs: 123-147to the axillary bud. The edited tobacco plants exhibit reduced suckergrowth compared to control tobacco plants.

Example 21 Development of Novel Mutations via Random Mutagenesis

Random mutagenesis of tobacco plants are performed using ethylmethanesulfonate (EMS) mutagenesis or fast neutron bombardment. EMSmutagenesis consists of chemically inducing random point mutations. Fastneutron mutagenesis consists of exposing seeds to neutron bombardmentwhich causes large deletions through double stranded DNA breakage.

For EMS mutagenesis, one gram (approximately 10,000 seeds) of Tennessee90 tobacco (TN90) seeds are washed in 0.1% Tween for fifteen minutes andthen soaked in 30 mL of ddH₂O for two hours. One hundred fifty (150) μLof 0.5% EMS (Sigma, Catalogue No. M-0880) is then mixed into theseed/ddH₂O solution and incubated for 8-12 hours (rotating at 30 R.P.M.)under a hood at room temperature (RT; approximately 20° C). The liquidthen is removed from the seeds and mixed into 1 M NaOH overnight fordecontamination and disposal. The seeds are then washed twice with 100mL ddH₂O for 2-4 hours. The washed seeds are then suspended in 0.1% agarsolution.

The EMS-treated seeds in the agar solution are evenly spread ontowater-soaked Carolina's Choice Tobacco Mix (Carolina Soil Company,Kinston, N.C.) in flats at ˜2000 seeds/flat. The flats are then coveredwith plastic wrap and placed in a growth chamber. Once the seedlingsemerge from the soil, the plastic wrap is punctured to allow humidity todecline gradually. The plastic wrap is completely removed after twoweeks. Flats are moved to a greenhouse and fertilized with NPKfertilizer. The seedlings are replugged into a float tray and grownuntil transplanting size. The plants are subsequently transplanted intoa field. During growth, the plants self-pollinate to form M1 seeds. Atthe mature stage, five capsules are harvested from each plant andindividual designations are given to the set of seeds from each plant.This forms the M1 population. A composite of M1 seed from each M0 plantare grown, and plants are phenotypically evaluated as described inExample 2. M1 plants exhibiting enhanced or reduced sucker growth areselected and screened for mutations using DNA sequencing and genemapping techniques known in the art.

Example 22 Regulating Sucker Growth Using Inducible Promoters

Inducible promoters are also used to express a functional gene in acontrolled manner to reduce or eliminate sucker development. Thesepromoters are induced by either a chemical spray or at certain timepoints (i.e., after topping). Exemplary promoters includealcohol-regulated promoters; tetracycline-regulated promoters;steroid-regulated promoters (e.g., glucocorticoid (See, for example,Schena et al., 1991, Proceedings of the National Academy of Sciences USA88:10421-10425, which is herein incorporated by reference in itsentirety); human estrogen; ecdysone); and metal-regulated promoters. Forexample, an RNase (e.g., SEQ ID NOs: 79 and 123-136), a VPE (e.g., SEQID NOs: 137-143), or a protease (e.g., SEQ ID NOs: 144-147) areexpressed in a tobacco plant with an inducible promoter.

In one example, a first vector containing a rat glucocorticoid receptorunder the control of a constitutive CsVMV promoter and a second vectorcontaining a sequence of interest (e.g., SEQ ID NOs: 83-101) operablylinked to one or more glucocorticoid response elements are createdaccording to Example 2. Modified tobacco plants are then generatedcontaining both of these vectors. Dexamethasone is sprayed on the plantsto induce the expression of the sequence of interest, then the plantsare phenotypically evaluated. The modified tobacco plants exhibitreduced sucker growth compared to control tobacco plants.

In a second example, a first vector containing a rat glucocorticoidreceptor under the control of Promoter P15 (SEQ ID NO: 117) and a secondvector containing a sequence of interest (e.g., SEQ ID NOs: 79 and123-147) operably linked to one or more glucocorticoid response elementsare created according to Example 2. Modified tobacco plants are thengenerated containing both of these vectors. Dexamethasone is sprayed onthe plants to induce the expression of the sequence of interest, thenthe plants are phenotypically evaluated. The modified tobacco plantsexhibit reduced sucker growth compared to control tobacco plants.

Example 23 Regulating Sucker Growth Using Phytotoxins and ImmuneReceptors

Tobacco plants are metabolically engineered to produce one or morephytotoxins (e.g., tabtoxin; coronatine; syringomycin; syringopeptin;phaseolotoxin) or immune receptors in an axillary meristem to inhibitcell growth or cell division within the axillary shoot meristem. See,for example, Bender et al., 1999, Microbiology and Molecular BiologyReviews 63:266-292, which is herein incorporated by reference in itsentirety. As an example, the tabA/tblA genes from Pseudomonas syringaerequired to produce tabtoxin are expressed with a tissue specificpromoter (e.g., SEQ ID NOs: 113-118, 148-160, and 204).

Immune receptor genes include wide range of genes. Some examples includeArabidopsis thaliana disease resistance protein RPSS (SEQ ID NO: 222)and tobacco TMV resistance N gene (SEQ ID NO: 224).

A transformation vector is created according to Example 2 to compriseSEQ ID NO: 221 driven by Promoter P15 (SEQ ID NO: 117). Modified tobaccoplants are then generated with a Promoter P15::RPS5 vector, and thenphenotypically evaluated, according to Example 2. The modified tobaccoplants exhibit reduced sucker growth compared to control tobacco plants.

Example 24 Regulating Sucker Growth Using Programmed Cell Death-InducingGenes

The presence of some proteins at elevated levels, in cells where theyare not normally expressed, or in subcellular locations where they arenot normally located, can induce cell death. Axillary bud specificpromoters, such as Promoter P1 (SEQ ID NO: 113) and Promoter P15 (SEQ IDNO: 117) are used to the express heterologous genes that in axillary budcells and ultimately result in death of the axillary bud.

Programmed cell death-inducing (PCD-inducing) enzymes (e.g.,transcription factors (SEQ ID NOs: 208, 210, and 212), kinases (SEQ IDNO: 214), cysteine proteases (SEQ ID NOs: 216 and 218), and caspases(SEQ ID NO: 220); see Table 6) are used to induce cellular death in theaxillary bud when their expression is driven by Promoter P15 (SEQ ID NO:117). Transformation vectors are created according to Example 2 tocomprise SEQ ID NOs: 208, 210, 212, 214, 216, 218, and 220 driven byPromoter P15 (SEQ ID NO: 117). Modified tobacco plants are thengenerated with the Promoter P15::PCD-inducing enzyme vector according toExample 2. Modified tobacco plants (T0 generation) and control tobaccoplants are grown to the layby stage, then plants are topped to removethe shoot apical meristem. Sucker growth is observed at the time oftopping, 24 hours after topping, and one week after topping. Expressionof PCD-inducing enzymes driven by Promoter P15 eliminates suckeroutgrowth in tobacco.

TABLE 6 Programmed Cell Death (PCD)-Inducing Genes SEQ ID NOs (NucleicAcid/Protein) Gene Name Species Sequence Description Sequence Source207/208 ALCATRAZ Arabidopsis Transcription factor Current Biologythaliana (myc/bHLH), fruit 2001, 11: 1941-1922 dehiscence 209/210 VND6Arabidopsis Transcription factor, JEB 2014, 65: 1313- thaliana PCD-xylogenesis 1321 211/212 VND7 Arabidopsis Transcription factor, JEB2014, 65: 1313- thaliana PCD- xylogenesis 1321 213/214 Adi3 Solanum AGCkinase, negative EMBO 2006, 25: 255- lycopersicum regulator, PCD with265 pathogen attack 215/216 XCP1 Arabidopsis Cysteine protease, PlantJournal 2008, thaliana PCD-xylogenesis 56: 303-315 217/218 XCP2Arabidopsis Cysteine protease, Plant Journal 2008, thalianaPCD-xylogenesis 56: 303-315 SlCysEP Solanum Cysteine protease, Planta2013, lycopersicum ricinosomal protease, 237: 664-679 PCD-endosperm219/220 Metacaspase Arabidopsis Protease, PCD during Plant Journal 2011,2d (ATMC4) thaliana biotic and abiotic 66: 969-982 stresses Caspase-likeSolanum PCD, like apoptosis in Planta 2000, protease lycopersicummammalian cells 211: 656-662

Example 25 Regulating Sucker Growth by Regulating microRNAs

miRNAs can be involved in the regulation of axillary bud development.See Ortiz-Morea et al., 2013, Journal of Experimental Botany64:2307-2320; and Wang et al., 2010, Molecular Plant 3:794-806, whichare herein incorporated by reference in their entireties. To identifymiRNAs involved in tobacco sucker development, total RNA samples areextracted from axillary buds and phloem of 4 week old TN90 tobaccoplants before topping and several time points after topping (e.g., 2hours after topping, 6 hours after topping, 24 hours after topping, 72hours after topping, 96 hours after topping). Small RNA (sRNA) areseparated and purified from the total RNAs. The resulting sRNA samples(three independently collected samples for each tissue type) areprocessed and subjected to Illumina sequencing. The Illumina reads aremapped and used to evaluate the expression profiles of miRNAs and othersmall RNAs (e.g., small-interfering RNAs (siRNAs), trans-acting siRNAs).Small RNAs, including miRNAs, that exhibit differential expression inaxillary buds before and after topping are identified. These sRNAs playa role in the formation or outgrowth of suckers. The identified sRNAs'precursor sequences and genomic sequences are subsequently identified.

Some tobacco sRNAs are associated with reduced sucker development and/orgrowth. Over-expression of these sRNAs is used to inhibit suckers. sRNAsfound to be associated with reduced sucker development and/or growth areplaced under the regulation of a promoter functional in an axillary bud(e.g., SEQ ID NOs: 113-118, 148-160, and 204), or a constitutivepromoter (e.g., CaMV 35S) according to Example 2.

A transformation vector is created according to Example 2 to comprisesRNAs of interest driven by Promoters P1, P11, P15, or PAB Thionin (SEQID NO: 118). Modified tobacco plants are then generated andphenotypically evaluated according to Example 2. The modified tobaccoplants exhibit reduced sucker growth compared to control tobacco plants.

miRNAs that promote sucker formation or outgrowth by repressing genesthat would otherwise inhibit sucker formation or outgrowth are targetedfor regulation by generating constructs comprising at least one miRNAdecoy under the regulation of a promoter functional in an axillary bud(e.g., SEQ ID NOs: 113-118, 148-160, and 204), or a constitutivepromoter according to Example 2.

A transformation vector is created according to Example 2 to comprise amiRNA decoy driven by Promoters P1, P11, P15, or PAB Thionin (SEQ ID NO:118). Modified tobacco plants are then generated and phenotypicallyevaluated, according to Example 2. The modified tobacco plants exhibitreduced sucker growth compared to control tobacco plants.

A further transformation vector is created according to Example 2 tocomprise a tobacco miR159 decoy driven by Promoter PAB Thionin (SEQ IDNO: 118). Tobacco mature miR159 (SEQ ID NO: 227) is complementary to atleast SEQ ID NOs: 1, 13, and 35, which all function to inhibit suckergrowth in tobacco. Preventing miR159-mediated degradation of SEQ ID NOs:1, 13, and 35 reduces sucker growth. Modified tobacco plants are thengenerated with an axillary meristem promoter::miR159 decoy vector, andthen phenotypically evaluated, according to Example 2. The modifiedtobacco plants exhibit reduced sucker growth compared to control tobaccoplants.

Example 26 Regulating Sucker Growth Via Modifying Auxin Synthesis andTransport

Removing the shoot apical meristem releases axillary buds from dormancyand promotes sucker outgrowth. Auxin derived from an intact shoot apicalmeristem suppresses sucker outgrowth. Typically, cytokinin induced byremoval of the shoot apical meristem promotes axillary meristemoutgrowth. Without being bound to any scientific theory, maintaining ahigh auxin:cytokinin ratio in and around axillary buds after removal ofthe shoot apical meristem can suppress axillary bud outgrowth.

Localized increases in auxin concentration in and around axillary budregions can suppress sucker outgrowth. Genes related to auxinbiosynthesis and/or transport are used to suppress axillary meristemoutgrowth when their expression is driven by Promoter P15 (SEQ ID NO:117) or other promoters functional in axillary buds (e.g., SEQ ID NOs:113-118, 148-160, 204, and fragments thereof). A transformation vectoris created according to Example 2 to comprise SEQ ID NO: 234 (YUCCA1;flavin monooxygenase) driven by Promoter P15 (SEQ ID NO: 117). Modifiedtobacco plants are then generated with the Promoter P15::YUCCA1 vectoraccording to Example 2. Modified tobacco plants (T0 generation) andcontrol tobacco plants are grown to the layby stage, then plants aretopped to remove the shoot apical meristem. Sucker growth is observed atthe time of topping, 24 hours after topping, one week after topping, twoweeks after topping, and three weeks after topping (FIGS. 19 and 20).Expression of SEQ ID NO: 234 driven by Promoter P15 suppresses suckeroutgrowth in tobacco.

Additional transformation vectors similar to Promoter P15::YUCCA1 arealso created using other auxin biosynthesis and auxin transport genesfrom Arabidopsis such as: PIN-FORMED1 (PIN1; SEQ ID NO: 236); TRYPTOPHANAMINOTRANSFERASE1/TRANSPORT INHIBITOR RESPONSE2 (TAA1/TIR2; SEQ ID NO:238); ALDEHYDE OXIDASE1 (AAO1; SEQ ID NO: 240); and INDOLE-3-ACETAMIDEHYDROLASE1 (AMI1; SEQ ID NO: 242). In addition to Promoter P15, severalpromoters functional in axillary buds (e.g., SEQ ID NOs: 113-118,148-160, 204, and fragments thereof) are used to drive the expression ofauxin biosynthesis and auxin transport genes (e.g., SEQ ID NOs: 234,236, 238, 240, and 242). Furthermore, tobacco genes homologous toArabidopsis auxin biosynthesis and auxin transport genes (e.g.,NtYUCCA-like, SEQ ID NOs:244 and 246; NtPIN1-like, SEQ ID NO:248;NtTAA1/NtTIR2-like, SEQ ID NO:250; NtAAO1-like, SEQ ID NO:252; andNtAM11-like, SEQ ID NO:254) are included in separate vector constructswith Promoter P15 as well as other promoters (e.g., SEQ ID NOs: 113-118,148, 160, 204, and fragments thereof) functional in axillary buds.

Each of these vectors is used to generate modified tobacco plants, andto then phenotypically evaluate the tobacco plants, as described inExample 2. The modified tobacco plants exhibit reduced sucker growthcompared to control tobacco plants.

TABLE 7 Auxin biosynthesis and auxin transport genes SEQ ID NOs (NucleicAcid/Protein) Gene Name Species Sequence Description Sequence Source234/235 YUCCA1 Arabidopsis FLAVIN Plant Cell 2007, thalianaMONOOXYGENASE 19: 2430-2439 236/237 PIN1 Arabidopsis PIN-FORMED1 Science1998, thaliana 282: 2226-2230 238/239 TAA1/TIR2 Arabidopsis TRYPTOPHANCell 1998, 133: thaliana AMINOTRANSFERASES 177-191; TRANSPORT INHIBITORPlant Physiology RESPONSE2 2009, 151: 168- 179 240/241 AAO1 ArabidopsisALDEHYDE OXIDASE1 J Biochem 1999, thaliana 126: 395-401 242/243 AMI1Arabidopsis INDOLE-3-ACETAMIDE FEBS J 2007, thaliana HYDROLASE 274:3440-3451

1.-24. (canceled)
 25. A method for producing a modified tobacco plantcomprising: (a) introducing a recombinant DNA construct comprising aheterologous promoter that is operably linked to a polynucleotideencoding a polypeptide having an amino acid sequence at least 90%identical or similar to SEQ ID NO: 233 to at least one tobacco cell; (b)selecting one or more tobacco cells from step (b) that comprise saidrecombinant DNA construct; and (c) generating one or more modifiedplants from the one or more plant cells selected in step (b).
 26. Themethod of claim 25, wherein said heterologous promoter is an axillarymeristem-specific promoter functional in a central zone, a peripheralzone, a rib zone, or any combination thereof.
 27. The method of claim25, wherein said heterologous promoter comprises a nucleic acid sequencehaving at least 90% sequence identity to a polynucleotide selected fromthe group consisting of SEQ ID NOs: 113, 115-118, 148, 149, 151, 153,155, 157, 159, and
 204. 28. The method of claim 25, wherein said one ormore modified plants comprise no suckers or reduced suckers as comparedto a control plant lacking said recombinant DNA construct when grownunder comparable conditions.
 29. The method of claim 28, wherein saidreduced suckers comprises reduced sucker mass, reduced sucker length, orboth.
 30. The method of claim 25, wherein said polypeptide has an aminoacid sequence 100% identical or similar to SEQ ID NO:
 233. 31. Themethod of claim 25, wherein said polypeptide is encoded by a nucleicacid sequence at least 95% identical to SEQ ID NO:
 232. 32. A method ofgrowing a modified tobacco plant comprising planting a modified tobaccoseed comprising a heterologous promoter that is operably linked to apolynucleotide encoding a polypeptide having an amino acid sequence atleast 90% identical or similar to SEQ ID NO:
 233. 33. The method ofclaim 32, wherein said heterologous promoter is an axillarymeristem-specific promoter functional in a central zone, a peripheralzone, a rib zone, or any combination thereof.
 34. The method of claim32, wherein said heterologous promoter comprises a nucleic acid sequencehaving at least 90% sequence identity to a polynucleotide selected fromthe group consisting of SEQ ID NOs: 113, 115-118, 148, 149, 151, 153,155, 157, 159, and
 204. 35. The method of claim 32, wherein said one ormore modified plants comprise no suckers or reduced suckers as comparedto a control plant lacking said recombinant DNA construct when grownunder comparable conditions.
 36. The method of claim 35, wherein saidreduced suckers comprises reduced sucker mass, reduced sucker length, orboth.
 37. The method of claim 32, wherein said polypeptide has an aminoacid sequence 100% identical or similar to SEQ ID NO:
 233. 38. Themethod of claim 32, wherein said polypeptide is encoded by a nucleicacid sequence at least 95% identical to SEQ ID NO:
 232. 39. A method forproducing a modified tobacco plant comprising: (a) crossing at least onetobacco plant of a first tobacco variety with at least one tobacco plantof a second tobacco variety, where the at least one tobacco plant of thefirst tobacco variety comprises a recombinant DNA construct comprising aheterologous promoter that is operably linked to a polynucleotideencoding a polypeptide having an amino acid sequence at least 90%identical or similar to SEQ ID NO: 233 to produce at least one progenytobacco plant; and (b) selecting at least one progeny tobacco plantproduced in step (a) that comprises said recombinant DNA construct. 40.The method of claim 39, wherein said heterologous promoter is anaxillary meristem-specific promoter functional in a central zone, aperipheral zone, a rib zone, or any combination thereof.
 41. The methodof claim 39, wherein said heterologous promoter comprises a nucleic acidsequence having at least 90% sequence identity to a polynucleotideselected from the group consisting of SEQ ID NOs: 113, 115-118, 148,149, 151, 153, 155, 157, 159, and
 204. 42. The method of claim 39,wherein said progeny tobacco plant comprise no suckers or reducedsuckers as compared to a control plant lacking said recombinant DNAconstruct when grown under comparable conditions.
 43. The method ofclaim 42, wherein said reduced suckers comprises reduced sucker mass,reduced sucker length, or both.
 44. The method of claim 39, wherein saidpolypeptide has an amino acid sequence 100% identical or similar to SEQID NO:
 233. 45. The method of claim 39, wherein said polypeptide isencoded by a nucleic acid sequence at least 95% identical to SEQ ID NO:232.